methyl cellulose method

Mouse Colony-Forming Cell AssaysUsing MethoCult®

TECHNICAL MANUALCatalog #28405

StemCell TechnologiesThe Cell Experts

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FOR RESEARCH USE ONLY

StemCell TechnologiesVersion 3.1.1

June 2005Catalog # 28405

In North AmericaTel: 604.877.0713 Fax: 604.877.0704Toll Free Tel: 1.800.667.0322Toll Free Fax: 1.800.567.2899e-mail: [email protected]: [email protected]

In the United KingdomTel: +44.(0).20.7537.7565 Fax: +44.(0).20.7515.5408Toll Free within United Kingdom:Tel: 0800.731.27.14 Fax: 0800.731.27.13e-mail: [email protected]

In EuropeTel: +33.(0).4.76.04.75.30Fax: +33.(0).4.76.18.99.63e-mail: [email protected]

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1.0 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2.0 StemCell Technologies’ Products for Mouse CFC Assays . . . . . . . . . . . . . . . . . . . . . . . . . 1

3.0 Storage and Handling of MethoCult® Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3.1 Thawing and Dispensing of Complete MethoCult® Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33.2 Thawing and Dispensing of Incomplete MethoCult® Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

4.0 Mouse Hematopoietic CFC Assays in MethoCult® Media . . . . . . . . . . . . . . . . . . . . . . . . . 5

4.1 Procedure Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54.2 Equipment and Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64.3 Mouse Cell Isolation Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

4.3.1 Bone Marrow Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64.3.2 Spleen Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74.3.3 Peripheral Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74.3.4 Fetal Liver, Day 10-16 PC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

4.4 Cell Counts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84.5 General Method for Set-up of CFC Assays in MethoCult® Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94.6 Mouse CFU-E Assay and Mature BFU-E Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104.7 Mouse BFU-E, CFU-GM, CFU-GEMM Assay, and CFU-GM Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104.8 Mouse CFU Pre-B Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

5.0 Descriptions and Photographs of Mouse Hematopoietic CFCs . . . . . . . . . . . . . . . . . . . . . 12

6.0 Frequently Asked Questions and Helpful Hints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

6.1 MethoCult® Media and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156.2 Preparation of Cell Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156.3 Set-up and Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156.4 Enumeration of Mouse CFC Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

7.0 Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

7.1 MethoCult® Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187.2 Preparation of Methylcellulose Medium with PWM-SCCM for BFU-E,

CFU-GM and CFU-GEMM assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187.3 Cloning of Cell Lines in Methylcellulose-Based Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Table of Contents

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1.0 Introduction

In the adult mouse bone marrow (BM), a small number of hematopoietic stem cells (HSCs) produce heterogeneous populations of activelydividing hematopoetic progenitors. These hematopoietic progenitors proliferate and differentiate resulting in the generation of millions ofmature blood cells daily. In vitro assay systems have been developed to quantify multi-potential progenitors and lineage-restricted progenitorsof the erythroid, granulocytic, monocyte-macrophage, and megakaryocyte-myelopoietic pathways as well as a subset of mouse pre-Blymphoid cells. When cultured in a suitable semi-solid matrix, individual progenitors called colony-forming cells (CFCs), proliferate to formdiscrete cell clusters or colonies. CFC assays are performed by placing a cell suspension into a semi-solid medium, such as methylcellulose,supplemented with nutrients and cytokines followed by incubation at 37°C for periods ranging from a few days to several weeks. The CFCs areclassified and enumerated based on the morphological recognition of one or more types of hematopoietic lineage cells within the colony. Thiscan be done in situ by light microscopy or after first staining the cells using cytochemical and immunocytochemical methods.

Various gelling agents including agar, agarose, methylcellulose, collagen and fibrin clots have been used for CFC assays. Methylcellulose is arelatively inert polymer that forms a stable gel with good optical clarity. It is commonly used at a final concentration of 0.9-1.2% in culturemedium supplemented with compounds including fetal bovine serum (FBS), bovine serum albumin (BSA), 2-mercaptoethanol, insulin,transferrin and recombinant cytokines or conditioned medium as a source of colony-stimulating factors. Methylcellulose-based medium permitsbetter growth of erythroid lineage cells than other types of semi-solid matrices, thus allowing the assay of erythroid, granulocyte, monocyteand multi-potential CFCs within the same culture.

2.0 StemCell Technologies’ Products for Mouse CFC Assays

MethoCult® Methylcellulose-Based MediumStemCell Technologies (StemCell) rigorously screens and selects components used in the manufacture of MethoCult® products. It is knownthat different batches of methylcellulose, fetal bovine serum (FBS) and bovine serum albumin (BSA) vary widely in their ability to promote CFCgrowth. If using media components other than those pre-screened and available from StemCell, it is important to test components individuallyand in combination for their ability to support the optimal growth and differentiation of hematopoietic cells.

Each batch of MethoCult® medium is manufactured using pre-tested components. All batches are performance tested using normalmouse BM and compared to a laboratory standard. Each batch is also sterility tested according to USP standards.

Complete MethoCult® media such as M3434, M3534 and M3630 are supplied at 100 mL per bottle (Catalog #03434, 03534, 03630).MethoCult® M3434 is also available as 3 mL per tube, 24 tubes per rack. (Catalog #03444). Other specialized formulations, which lackcomponents such as recombinant growth factors, are available to meet the specific requirements of investigators (Catalog #03231, 03334,03234, 03236, 03134; See Table 1 and Appendices 7.1 and 7.2). Custom formulations are also available. Please contact StemCellTechnologies for more information.

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Catalog # Contains Applications

0343403444

rm SCF, rm IL-3, rh IL-6, rh Epo

Detection of BFU-E, CFU-GM, CFU-GEMM in BM, spleen, PB and fetal liver

03534 rm SCF, rm IL-3, rh IL-6 Detection of CFU-GM in BM, spleen, PB and fetal liver

03630 rh IL-7 Detection of CFU-pre-B in BM

03334 rh Epo Detection of CFU-E, mature BFU-E

03234 Without cytokines Allows researchers to add cytokines of their choice for applications including:- drug toxicity testing- detection of specific hematopoietic progenitors (e.g. CFU-Mast with rm SCF)- investigate action of novel factors- hematopoietic colony assays in other species- selection of transduced hematopoietic progenitors- cloning and selection of non-adherent cell lines

03231 Without cytokines

03236Serum-freewithout cytokines

Allows researchers to add cytokines of their choice in defined serum-free methylcellulose-based medium

03134 Methylcellulose base Preparation of methylcellulose-based medium for specialized applications

MegaCult®-CCollagen-based mediumwithout cytokines

Detection of CFU-Mk in BM and other hematopoietic tissues.See our website or contact your StemCell Technical Representative.

Catalog # Description

02715, 02915 rm G-CSF. For growth of granulocytic progenitors

02732, 02932 rm GM-CSF. For growth of granulocytic and monocytic progenitors

02733, 02903 rm IL-3. Used in combination with other cytokines to promote growth of early myeloid progenitors of all lineages

02751, 02951 rm M-CSF. For growth of monocytic progenitors

02731, 02931rm SCF. For growth of mast cells and used in combination with other cytokines to promote growth of myeloid andlymphoid progenitors

02720, 0292002620, 02820

rm TPO or rh TPO. Used in combination with other cytokines to promote growth of megakaryocytic progenitors

02625 rh EPO. Used in combination with other cytokines for growth of erythroid progenitors

02100 PWM-SCCM: Pokeweed Mitogen Spleen Cell Conditioned Medium. Used as source of colony stimulating factors

Additional CytokinesRefer to StemCell Technologies’ Catalog, website or contact your Technical Representativefor a complete list of available cytokines.

Table 1: MethoCult® Products and Applications

Table 2: Recombinant Cytokines for Culture of Mouse Hematopoietic Cells

BM: Bone Marrow; PB: Peripheral Blood

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3.0 Storage and Handling of MethoCult® Media

MethoCult® media should be stored at -20°C. Store for up to one month at 2-8°C. Repeated freezing and thawing is not recommended.

3.1 Thawing and Dispensing of Complete MethoCult® Media

Complete MethoCult® media (Catalog #03434, 03534, 03630) is supplied at 100 mL per bottle. It is formulated to allow the addition of cells tothe MethoCult® product at a 1:10 (v/v) ratio.

1. Thaw MethoCult® medium overnight under refrigeration or at room temperature.2. Mix contents of bottle by shaking vigorously for 30-60 seconds.3. Let stand for 5 minutes to allow bubbles to rise.4. To dispense MethoCult® medium into tubes, use a 6 or 12 mL sterile luer-lock syringe and 16 gauge blunt-end needle.

Blunt-end needles should be used for safety reasons and to facilitate the accurate dispensing of methylcellulose-based medium.The use of 16 gauge blunt-end needles prevents injury due to needle pricks. Methylcellulose is a viscous solution and cannot beaccurately dispensed using pipettes due to adherence of the medium to the inside of the pipette.

5. To remove the air from the syringe, place needle below surface of the MethoCult® medium and draw up approximately 1 mL.Gently depress plunger and expel medium completely. Repeat until no air space is visible.

Catalog # Description

06200, 06250Fetal Bovine Serum. Prescreened and selected for growth of mouse hematopoietic cells insuspension or methylcellulose-based cultures.

07050 Trypan Blue. For cell viability counts

07060 3% Acetic Acid with Methylene Blue. For nucleated cell counts

07700 Iscove's MDM with 2% FBS. For cell washing

07800, 07850 Ammonium Chloride. For lysis of red blood cells

09300 Detoxified 10% BSA Solution (in Iscove’s MDM)

09500BIT 9500 (5X media supplement containing BSA, transferrin and insulin). Prescreened andselected for growth of mouse hematopoietic cells in suspension or methylcellulose-based cultures.

09600, 09650 StemSpan® SFEM. For growth of hematopoietic cells in suspension cultures.

27100, 27150 Pre-tested 35 mm culture dishes

27305 Cell strainer, 40 µm. For peparation of single cell suspensions

27500 Gridded Scoring Dishes

28110, 28120 Blunt-End Needles. For dispensing of methylcellulose-based medium.







Table 3: Support Products for Mouse Cells

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* Cells are added in 0.3 mL volume to 3.0 mL MethoCult® for duplicate cultures and 0.4 mL to 4.0 mL MethoCult® for triplicate cultures.** (i.e. Cytokines, IMDM)

*** (i.e. FBS, BSA, Cytokines, IMDM)

MethoCult® Catalog #

03434, 03534, 03630 03334 03234, 03231, 03236 03134

MethoCult® volume per bottle 100 mL 90 mL 80 mL 40 mL

Additional volume required for100 mL total volume

0 mL 10 mL 20 mL 60 mL

Volume dispensed per tube forduplicate* 1.1 mL cultures(volume of additional components)

3.0 mL

(0 mL)

2.7 mL

(0.3 mL)**

2.4 mL

(0.6 mL)**

1.2 mL

(1.8 mL)***

Volume dispensed per tube fortriplicate* 1.1 mL cultures(volume of additional components)

4.0 mL

(0 mL)

3.6 mL

(0.4 mL)**

3.2 mL

(0.8 mL)**

1.6 mL

(2.4 mL)***

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6. Draw up methylcellulose medium into syringe and dispense desired volume into 14 mL sterile culture tubes. Complete MethoCult®

media (Catalog #03434, 03534, 03630) is formulated to allow the addition of cells at a 1:10 (v/v) ratio.• Dispense 3 mL per tube to yield duplicate cultures of 1.1 mL each.• Dispense 4 mL per tube to yield triplicate cultures of 1.1 mL each, etc.

It is preferable to dispense the entire contents of the bottle into tubes containing the appropriate volume to avoid repeatedfreezing and thawing of the bottle.

7. Cap tubes tightly. Store at -20°C or for one month at 2-8°C until use.

3.2 Thawing and Dispensing of Incomplete MethoCult® Media

'Incomplete' MethoCult® media (Catalog #03134, 03231, 03234, 03236, 03334; refer to Appendix 7.1, Table 8) are provided to allowresearchers to add components to prepare formulations for specific cell culture requirements. Components should be added to MethoCult®

bottles to yield a total volume of 100 mL and then dispensed into tubes. Alternatively, appropriate volumes can be dispensed into tubes, frozenand desired components added at time of use. It is important to dilute MethoCult® as described to maintain optimal viscosity of themethylcellulose-based medium.

1. Thaw MethoCult® medium overnight under refrigeration or at room temperature.2. Add components, if desired (See Table 4). Mix contents of bottle of by shaking vigorously for 30-60 seconds.3. Let stand for 5 minutes to allow bubbles to rise.4. To dispense MethoCult® medium into tubes, use a 3, 6 or 12 mL sterile luer-lock syringe and 16 gauge blunt-end needle (as

described in Section 3.1).5. Cap tubes tightly. Store at -20°C or for one month at 2-8°C until use.

Table 4: Preparation of MethoCult® Media

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4.0 Mouse Hematopoietic CFC Assays in MethoCult® Media

4.1 Procedure Diagram

Prepare CellsProcess mouse cells by:• ammonium chloride lysis• density gradient separation• progenitor cell enrichment with

EasySep®, StemSep®, SpinSep® orFACS

Wash cells (e.g. in Iscove’s MDM plus2% FBS), then count and adjust cellconcentration.

1step

Add Cells to MethoCult®

Add cells to MethoCult® and vortex.Let tube stand to allow bubbles to dissipate.

2step

Plate and IncubateDispense cells into pre-tested culture dishesusing syringe and blunt-end needle.Incubate mouse cells for 7-14 days inhumidified incubator at 37°C and 5% CO2.

3step

Count ColoniesCount and evaluate colony types usinginverted microscope and gridded scoringdishes. Alternatively, individual coloniesmay be plucked for routine staining, PCR,or cytogenetic analysis.

4step

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4.2 Equipment and Reagents Required

• Biohazard Safety Cabinet approved for Level II handling of biological material• Incubator set at 37°C with 5% CO2 in air and >95% humidity

Use of water-jacketed incubators with a water pan placed in the chamber is recommended.• Inverted Microscope

Use of a quality inverted microscope equipped with a 10 or 12.5X eyepiece objective and2X, 4X and 10X planar objectives is recommended.

• Laboratory centrifuge• Vortex• Micropipettors with sterile pipette tips• Automated cell counter or Neubauer hemacytometer• Sterile pipettes• Sterile test tubes• Sets of sterile sharp fine scissors and forceps should be reserved for animal dissection.

4.3 Mouse Cell Isolation Procedures

Laboratory mouse strains are routinely used between 6-12 weeks of age. For younger or older animals, transgenic mouse strains andcompound-treated mice, it is important to use strain and age matched controls. Mice are maintained and sacrificed using protocols andprocedures approved by the institution. Numbers of nucleated cells isolated from the bone marrow and spleen may vary depending on the ageof the animal. Unless individual animals are being evaluated, the cells from 2 or more mice should be pooled.

4.3.1 Bone Marrow Cells

Sacrifice mouse using procedures recommended by institution.1. Position mouse on its back and wet fur thoroughly with 70% isopropyl alcohol. This step decreases the possibility of contaminating

cell preparations with fur.2. Cut a slit in the fur just below the rib cage without cutting the peritoneal membrane. Non-sterile scissors can be used for this step.3. Firmly grasp skin and peel back to expose hind limbs.4. Using sterile sharp dissecting scissors, cut the knee joint in the center. Cut through ligaments and excess tissue.

Use of sharp scissors will prevent splitting of the bone.5. Grasp femur with forceps and cut femur near hip joint.6. Free tibia by cutting near the ankle joint.7. Trim the ends of the long bones to expose the interior marrow shaft. Put bones in sterile petri dish or in sterile culture medium and

place on ice. Bones can be collected from multiple animals.8. Using a 3 cc syringe with a 21 gauge (g) needle, draw up 1-3 mL of cold medium containing 2% FBS. A smaller needle (22g or 23g)

may be required to remove cells from the tibia.Cells should be isolated as soon as possible after animal is sacrificed.

9. Insert bevel of needle into marrow shaft and flush marrow into sterile 14 mL culture tube. The bone should appear white once all themarrow has been expelled. Repeat same procedure with all bones. The same medium can be used to isolate bones from 1-3 animals.Suitable culture medium includes Iscove's MDM, Alpha MEM or PBS. Cells should be isolated in small volumes of medium.

10.To make a single cell suspension, gently draw medium and cells up and down with a 3 cc syringe and 21 g needle. Repeat 3-4 times,keeping needle below medium surface.

11.Keep the cells in medium on ice until use.

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4.3.2 Spleen Cells

Sacrifice mouse using procedures recommended by institution.1. Position mouse (right side down) and wet fur thoroughly with 70% isopropyl alcohol.2. Make an incision (~1 cm) in the fur just under the rib cage. Avoid cutting peritoneal membrane.3. Peel back skin to expose the peritoneum. The spleen, a dark red coloured organ, should be visible.4. Using sterile dissecting scissors make an incision in the peritoneal membrane.5. To avoid crushing or tearing of spleen, grasp the fatty tissue attached to the spleen using blunt-end forceps and gently pull upward.

Cut splenic blood vessel to detach spleen. Place spleen in sterile petri dish on ice or in cold sterile culture medium.Suitable culture medium includes Iscove's MDM, Alpha MEM or PBS.

6. To prepare a single cell suspension, place 1-3 spleens on a nylon mesh cell strainer (Catalog #27305) within a 35 mm dish.7. Mince spleen using fine sterile scissors.8. Place strainer in 50 mL Falcon tube containing 2-3 mL of medium with 2% FBS or add 2-3 mL of medium to 35 mm dish. Gently

press tissue through strainer using the end of a sterile plunger from a 3 mL syringe into the medium.9. Rinse cells in strainer using an additional small volume of medium.

10. Cell aggregates can be disrupted by drawing cell suspension up and down 3-4 times using a 3 cc syringe and 21g needle if required.11. Place cells in 14 mL sterile culture tube and let stand for 3-5 minutes to allow tissue fragments to settle. Transfer cell suspension to

new sterile culture tube.12. If desired, wash cell suspension twice and resuspend in a small volume of medium.

4.3.3 Peripheral Blood

Sacrifice mouse using procedures recommended by institution. The method of choice for isolating peripheral blood will depend on theprotocols approved by individual institutions. Isolation of PB from mice sacrificed by CO2 gassing is described below.

1. Attach a 21g needle to a 1 cc sterile syringe. Draw up small volume of anticoagulant solution (i.e. heparin 100 IU/mL) wetting interiorof syringe and expel solution leaving 20 -50 µL of anticoagulant in needle and syringe.

2. Sacrifice animal by C02 asphyxiation. Working as quickly as possible, open the chest cavity.3. Insert needle into ventricle and slowly draw blood into the syringe. Usually 0.5-1 mL of blood can be obtained by this method.4. Place peripheral blood in a 14 mL culture tube containing sufficient heparin to give final concentration of 15-20 IU/mL. Add 10 times

volume of ammonium chloride solution (Catalog #07800, 07850). Cap tightly and mix by inverting tube 3-4 times.5. Incubate on ice for 5-15 minutes, mixing several times.6. Centrifuge for 7 minutes at 1200 rpm (400 x g). Carefully decant or aspirate off the ammonium chloride solution.7. Resuspend cells in small volume of medium using sterile pipette.

Suitable culture medium includes Iscove's MDM, Alpha MEM or PBS.8. Wash cells twice using Iscove's MDM with 2% FBS and resuspend in a small volume of medium.

4.3.4 Fetal Liver, Day 10-16 PC

Sacrifice pregnant female mouse using procedures recommended by institution.1. Wet fur with 70% isopropyl alcohol and make an ~1 cm incision in the pelt taking care not to cut peritoneal membrane. Peel back

the pelt.2. Remove placenta-fetuses from animal and place in sterile petri dish on ice. If there will be a delay in isolating cells, place

placenta-fetuses in culture medium in sterile 50 mL tube.Suitable culture medium includes Iscove's MDM, Alpha MEM or PBS.

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3. Place placenta-fetuses in 100 mm petri dish with 5 mL of cold Iscove's MDM with 2% FBS. Using sterile fine scissors and forceps,isolate individual fetus and place in new petri dish with medium.

4. To isolate the fetal liver, place fetus in new 60 mm petri dish (no medium). Isolate fetal livers and transfer to new 60 mm dishcontaining ~1 mL of medium.

5. Mince fetal livers finely using scissors. Add 1-2 mL of medium. Cell aggregates can be disrupted by drawing cell suspension up anddown 3- 4 times using an 3 cc syringe and 21g needle.

6. Place cells in 14 mL sterile culture tube and let stand for 3-5 minutes to allow tissue fragments to settle. Transfer cell suspension tonew sterile culture tube. Fill tube with medium and centrifuge for 7-10 min at 1200 rpm (400 x g). Resuspend cells in small volume ofmedium and mix using sterile pipette.In normal day 14.5 fetal livers, >80% of the cells are nucleated erythroid precursors which are not removed by ammonium chloridelysis.

7. Wash cells twice using Iscove's MDM with 2% FBS and resuspend in small volume of medium.

4.4 Cell Counts

1. To perform a manual nucleated cell count, first dilute the cells in 3% acetic acid with methylene blue (Catalog #07060).Recommended dilution for bone marrow and spleen cells is 1/50 to 1/100; for peripheral blood cells, dilute cells to 1/20. Example: Fora 1/50 dilution, use a micropipettor with sterile tips and add 20 µL of cells to 980 µL of 3% acetic acid with methylene blue.

2. Prepare the hemacytometer by first cleaning the chambers with alcohol and then wiping dry.3. Carefully position the coverslip over both chambers.4. Mix the diluted cells well. Fill both chambers of the hemacytometer using a micropipettor or a capillary tube.

Do not over- or underfill the chambers.5. Count total nuclei in 4 large squares (1 x 1 x 0.1 mm) or ≥100 cells.6. Determine the cell count (cells per mL) as follows:

AVERAGE CELL COUNT PER SQUARE x DILUTIONFACTOR x 104 = CELL COUNT PER mL

Figure 1. Neubauer hemacytometershowing dimensions of each square.

Depth = 0.1 mm

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4.5 General Method for Set-up of CFC Assays in MethoCult® Media

1. Labels lids of 35 mm culture dishes at the edge using a permanent fine felt marker.2. Thaw tubes of MethoCult® medium overnight under refrigeration or at room temperature.

If required, add additional components. Refer to page 8, Section 3.2 for details.3. Vortex tubes to ensure all components are thoroughly mixed.4. Prepare cells at 10X the final concentration required.

Example: To achieve 1 x 105 cells per dish, a cell suspension of 1x106 cells per mL is prepared.5. Add 0.3 mL of cells to 3 mL of MethoCult® medium for duplicate cultures or 0.4 mL of cells to 4 mL of MethoCult® medium for triplicate

cultures.6. Vortex tubes to ensure all cells and components are thoroughly mixed.7. Let tube stand for 5 minutes to allow bubbles to dissipate.8. To dispense MethoCult® medium into culture dishes, attach 16 gauge blunt-end needle to a 3 cc syringe.9. To expel most of the air from the syringe, place needle below surface of solution and draw up approximately 1 mL. Gently depress the

plunger and expel medium completely. Repeat until no air space is visible.10. Draw up methylcellulose medium into syringe. Dispense 1.1 mL per 35 mm dish.

Culture dishes (Catalog #27100, 27150) for CFC assays are pretested for minimal cell adherence. Adherence of cells during culturecan cause inhibition of colony growth and obscure visualization of colonies.

11. Distribute methylcellulose medium evenly by gently tilting and rotating each dish.12. Place the two dishes into a 100 mm petri dish. Add a third, uncovered 35 mm dish containing 3 mL of sterile water. Replace lid of

100 mm petri dish.The use of a 100 mm petri dish and water dish helps maintain humidity and minimize contamination during culture and handling.

13. Place cultures in an incubator maintained at 37°C, 5% CO2 in air and ≥95% humidity.Culture conditions are very important to ensure optimal hematopoietic colony growth. We recommend the use of a water-jacketedincubator with an open pan of water placed in the incubator chamber. A suitable additive (i.e. copper sulfate crystals) can be added tothe water in pan to inhibit microbial growth. Incubator temperature should be confirmed using a thermometer placed in the incubatorchamber and CO2 levels should be routinely monitored using a fyrite CO2 device.

Tissue Type Total Cells

Femur 1-2 x 107

Tibia 0.6-1 x 107

Spleen 1-2 x 108

Peripheral Blood, nucleated cells per mL 3-5 x 106

Day 14.5 PC Fetal Liver 1 x 107

Table 5: Expected Numbers of Nucleated Cells

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4.6 Mouse CFU-E and Mature BFU-E Assays1. Add Iscove's MDM with 2% FBS at a 1:9 ratio to M3334 (10 mL to 90 mL bottle; 0.3 mL to 2.7 mL for duplicate cultures; 0.4 mL to

3.6 mL for triplicate cultures).2. Isolate mouse BM cells as described in Section 4.3.1 and dilute to 2 x 106/mL in Iscove's MDM with 2% FBS.

Lysis of red blood cells in mouse BM cell suspension is not required.3. Set-up CFC assay as described in Section 4.5. The plating concentration will be 2 x 105 BM cells per 35 mm dish.4. Incubate for 2 days at 37°C with 5% CO2 and ≥95% humidity for CFU-E or 3-4 days for mature BFU-E.5. Identify and count colonies as described in Section 5.0.

Table 6: Expected CFU-E and Mature BFU-E Numbers in Mouse BMRepresentative values for C57BL/6 mice at 8-12 weeks of age.

4.7 Mouse BFU-E, CFU-GM and CFU-GEMM Assay, and CFU-GM Assay1. Thaw tubes of MethoCult® M3434 or M3534 (Catalog #03434, 03534, for CFU-GM only) overnight under refrigeration (2-8°C) or at

room temperature.2. Isolate mouse BM, blood, spleen or fetal liver cells as described in Section 4.3.3. Dilute cells in Iscove's MDM with 2% FBS at 10X final concentration required (see Table 7).

Lysis of red blood cells in mouse BM cell suspension is not required. Lysis of mature red blood cells in spleen and blood samples isrecommended. If the expected number of colonies cannot be estimated, set-up duplicate cultures at 2-3 different cell concentrations.

4. Set-up CFC assay as described in Section 4.5.5. Incubate for 12 days at 37°C with 5% CO2 and ≥95% humidity. If CFCs cannot be counted on day 12, transfer cultures to incubator

maintained at 33°C, 5% CO2 in air and ≥95% humidity and count as soon as possible. If desired, BFU-E can be counted at day 7-10of culture.

6. Identify and count colonies as described in Section 5.0.

ProgenitorCells per 35 mm Dish

(1.1 mL Cultures)Colonies per Culture

CFU-E 2 x 105 340 ± 80

Mature BFU-E 2 x 105 50 ± 12

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* Researchers should perform preliminary experiments to determine CFC numbers in control mice. Analysis of these tissues (spleen, blood) ismost often performed for comparative analysis (e.g. pre- and post-drug or cytokine administration, CFC numbers in homozygous (-/-), andheterozygous (+/-) transgenic mice, and control (+/+) littermates).

4.8 Mouse CFU Pre-B Assay

1. Thaw tubes of MethoCult® M3630 (Catalog #03630) overnight under refrigeration (2 - 8°C) or at room temperature.2. Isolate mouse BM cells as described in Section 4.3.1 and adjust concentration to 5 x 105 or 1 x 106 cells per mL. The plating

concentration will be 5 x 104 or 1 x 105 cells per 35 mm dish.The numbers of CFU pre-B may vary between mouse strains and with age. If the expected number of colonies cannot be estimated,set-up duplicate cultures at 2-3 different cell concentrations.

3. Set-up CFC assay as described in Section 4.5.4. Incubate for 7 days at 37°C with 5% CO2 and ≥95% humidity.5. Identify and count colonies as described in Section 5.0.

Expected CFU-pre B numbers: 46 ± 6 per 5 x 104 BM cells.

Addition of 5-20 ng/mL recombinant mouse SCF to MethoCult® 03630 may increase the numbers of CFU-pre-B detected. However,addition of rm SCF also promotes myeloid growth within the cultures which can inhibit the growth of CFU-pre-B. The numbers ofmyeloid CFC also increases when higher input cell numbers are used due to endogenous cytokine production. It is recommendedthat preliminary experiments using 2-3 different cell densities (i.e. 0.5, 1, 2 x 105 per culture), and 10 ng/mL IL-7 in the presence andabsence of rm SCF, be performed to establish optimal conditions for your research application.

ProgenitorColonies per Culture

Representative values for C57BL/6 mice at 8-12 weeks of ageand day 14.5 post coitus (PC) fetal liver.

Bone Marrow, 2 x 104 cells plated per 35 mm dish

BFU-E 8 ± 3

CFU-GM 64 ± 16

CFU-GEMM 3 ± 1

Spleen, 1 x 105 cells plated per 35 mm dish Low numbers of CFCs are expectedin normal adult mice spleen and blood*Blood, 1 x 105 cells plated per 35 mm dish

Day 14.5 PC Fetal Liver, 2 x 104 cells plated per 35 mm dish

BFU-E 9 ± 3

CFU-GM 55 ± 10

CFU-GEMM 3 ± 2

Table 7: Expected BFU-E, CFU-GM and CFU-GEMM Numbers

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5.0 Descriptions and Photographs of Mouse Hematopoietic CFCs

Mouse hematopoietic progenitors can be quantitated in cell suspensions of adult bone marrow, spleen, and peripheral blood and duringdevelopment in tissues such as fetal liver. The classes of mouse hematopoietic progenitors detected using MethoCult® media include:

CFU-E: Colony-forming unit-erythroid. These are mature erythroid progenitors that form 1-2 clusters of maturing erythroblasts in the presenceof erythropoietin (EPO). CFU-E are counted after 2-3 days of culture. Description: These colonies are very tiny as seen under 40-50Xmagnification. One cluster contains at least 8 (~8-32) erythroblast cells. Erythroblast cells within the cluster are irregular in shape and appearfused together. There is no picture shown, however a CFU-E looks like a single cluster of the BFU-E colony (contains approximately 250clusters) as seen in Photo 1.

Mature BFU-E: Mature burst-forming unit-erythroid. The mature BFU-E form small colonies containing 3 or more clusters of erythroid cells orsingle larger colonies in the presence of EPO only. Mature BFU-E are generally counted after 3-4 days of culture. Description: Looks like agroup of 3 clusters of the BFU-E colony (contains approximately 250 clusters) as seen in Photo 1.

BFU-E: Burst-forming unit-erythroid. BFU-E require EPO and cytokines with burst-promoting activity such as Interleukin-3 (IL-3) and Stem CellFactor (SCF) for optimal growth. BFU-E are enumerated after 7-14 days of culture. Description: Made up of erythroid clusters and minimumof 30 cells. Each individual cluster contains a group of cells that are tiny, irregular in shape and difficult to distinguish. The cells appear fusedtogether. BFU-E do not usually have a dense core and the clusters are relatively scattered. However, it is best to confirm BFU-E by looking atthe individual clusters within each colony. Refer to Photos 1 and 2.

CFU-GM: This classification includes CFU-granulocyte (CFU-G), CFU-macrophage (CFU-M), and CFU-granulocyte macrophage (CFU-GM).The colonies contain 30 to thousands of granulocytes (CFU-G), macrophages (CFU-M) or both cell types (CFU-GM). Description: Made up ofat least 30 cells per colony. CFU-GM colonies often contain multiple cell clusters (dense core surrounded by cells). The monocytic lineagecells are large cells with an oval to round shape and appear to have a grainy or grey centre (see Photos 4, 5, 7 and 8). The granulocyticlineage cells are round, bright, and are much smaller and more uniform in size than macrophage cells (see Photos 2, 3, and 6). It is easy tosee individual cells of a CFU-GM colony, especially in the periphery of the colony, as seen in Photos 3 and 6.

CFU-GEMM: CFU-granulocyte, erythroid, macrophage, megakaryocyte. CFU-GEMM are multi-potential progenitors that require EPO and twoor more cytokines to support the growth and differentiation of lineage-committed daughter cells within the forming colony. Because of theirprimitive nature, CFU-GEMM tends to produce large colonies of >500 cells containing erythroblasts and recognizable cells of at least twoother lineages. Description: CFU-GEMM colonies are generally large (>500 cells per colony) and have a highly dense core with an indistinctborder between the core and peripheral cells. Erythroblast clusters should be visible along the periphery of the CFU-GEMM colony. Monocyticand granulocytic cells (see CFU-GM) should be easily identifiable and clusters of large megakaryocytic cells are usually seen. Refer to Photos9 and 10.

CFU pre-B: A subset of B-lymphoid progenitors can be detected in the presence of Interleukin-7 (IL-7). Description: Made up of at least 30cells. Although CFU pre-B colonies vary in size and morphology, individual cells appear tiny and irregular to oval in shape. Some CFU pre-Bcolonies are very dense with very few cells in the periphery and some have a smaller core with more cells in the periphery. Refer to Photos 11and 12.

CFU-Mk: CFU-megakaryocyte. Although megakaryocytic progenitors can be cultured in methylcellulose-based medium, it can be difficult todistinguish CFU-Mk based on colony morphology. Therefore, we recommend that CFU-Mk be enumerated in collagen-based, MegaCult®-Cfollowing staining of megakaryocytes in dehydrated gels by esterase staining. The MegaCult®-C Technical Manual is available on our websiteat www.stemcell.com. No picture of CFU-Mk shown.







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BFU-E 125X BFU-E CFU-GM 125X

CFU-G CFU-M 125X

CFU-GM 50X

CFU-GM BFU-E 50X

CFU-M 125X

Photo 1 Photo 2

Photo 3 Photo 4

Photo 6Photo 5

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CFU-G CFU-M 125X CFU-M CFU-GEMM 50X

CFU-GEMM 125X

CFU Pre-B 125X

CFU-GEMM 50X

CFU Pre-B 50X

Photo 7 Photo 8

Photo 10Photo 9

Photo 11 Photo 12

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6.0 Frequenty Asked Questions and Helpful Hints

6.1 MethoCult® Media and Reagents

Why should MethoCult® methylcellulose-based media be thawed at room temperature or in the refrigerator instead of at 37°C?• The methylcellulose will not be homogenous in frozen MethoCult® products and small ‘lumps’ may be present if the product is

thawed rapidly at 37°C. If the product is inadvertently thawed at 37°C, place the bottle on ice for a 1-2 hours or in the refrigeratorfor 2-3 hours (the ‘lumps’ will not dissolve at 37°C!). Shake the bottle vigorously for 30-60 seconds before dispensing.

6.2 Preparation of Cell Samples

Is it necessary to eliminate red blood cells (RBC) from the cell suspensions?• It is usually not necessary to lyse or remove RBC in normal bone marrow cell suspensions. The low numbers of RBC that will be

present in the CFC cultures do not make the colonies difficult to see or inhibit colony growth.• Lysis of RBC in peripheral blood (PB) samples by ammonium chloride treatment is necessary as the large number of RBC present

in PB samples can obscure the colonies and make the colonies difficult to see or inhibit colony growth.• Day 10-16 PC fetal liver contains a high percentage of nucleated erythroid cells (greater than 80%). BFU-E, CFU-GM, CFU-GEMM

analysis can be done on unseparated fetal liver cell suspensions. If desired, erythroid cells can be removed by density gradientcentrifugation or by depletion of TER119+ cells.

Should I do viable cell counts or total nucleated cell counts on mouse cell samples?• Nucleated cells counts using 3% acetic acid (Catalog #07060) are routinely performed as cell viability should be high in most

freshly isolated cell suspensions.

6.3 Set-up and Culture

Why should pre-tested petri dishes be used for CFC assays?• Use of culture-treated dishes or some brands of petri dishes may promote the adherence of bone marrow stromal cells, fibroblasts

and monocytes. Their presence can inhibit progenitor growth and make it difficult to count and distinguish the types of CFCpresent. Use of our pre-tested culture dishes (Catalog #27100, 27150) is recommended.

How do I estimate the number of cells to place in CFC assays?• Suggested cell numbers to be plated in the CFC assays are shown in Table 7. When optimal plating cell concentrations cannot be

estimated (i.e. transgenic or drug-treated mice in which hematopoiesis may be perturbed) set up 2-3 cell concentrations that differby 2-4 fold. Choose a cell concentration that results in ~40-120 colonies per 1.1 mL culture.

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Why is the number of colonies lower than expected?Possible reasons:• Errors in cell counts or cell dilutions resulting in too few cells being set-up in the CFC assay.• Contamination of cultures by bacteria, yeast or fungi. Bacterial contamination often results in the medium having a milky, orange

colour. Contamination is often caused by lack of good sterile technique or contaminated reagents. If contamination occurs, be sureto discard all contaminated cultures, opened bottles of medium used for cell processing and sanitize the incubator usingrecommended procedures.

• Improper incubator conditions. Dehydration of cultures can occur if high humidity (≥95%) is not maintained over the culture period.Use of small chamber water-jacketed incubators, a water pan in incubator chamber and dishes containing sterile water (seeSection 4.5) is recommended. The incubator temperature and CO2 levels should be routinely monitored using a thermometerplaced within the chamber and a fyrite analyser, respectively. No or low CO2 levels result in the medium having a purple colour. It isimportant to use medical grade CO2 for incubators as colony growth inhibition by unknown contaminants in the gas source hasbeen reported.

• MethoCult® medium has expired or has not been stored properly.• Loss of progenitors in the cell suspension. The CFC assays should be set-up as soon as possible following isolation of the cells, as

prolonged storage may result in loss of progenitors. If cells are cryopreserved or stored for times greater than ~6-8 hours, suitablecontrols should be included in your experiments.

Can I add antibiotics or other drugs to MethoCult®?• Antibiotics, drugs and other components can be added to the medium before the addition of cells. One important consideration is to

add all components in volumes that will maintain the correct viscosity of the MethoCult® medium. Drugs, cells and components areadded in a 1:10 v/v ratio to the incomplete methylcellulose formulations, as described in Table 4, page 8. To add components tocomplete, ready-to-use formulations such as M3434, M3534 and M3630, it is necessary to add the cells in a smaller volume.Example: To a 3 mL tube of M3434, add the desired component in a 0.1 mL volume, vortex to mix and then add the appropriatecell numbers in a 0.2 mL volume to maintain the correct 1:10 v/v ratio.

Is it necessary to add antibiotics to the media?• Addition of antibiotics should not be required if sterile reagents, certified biosafety cabinets and good aseptic technique are used. If

necessary, Penicillin (final 100 units/mL) and Streptomycin (final 100 µg/mL) can be included. Anti-fungal agents like amphotericinB can be also potentially be used, but preliminary experiments (to evaluate CFC growth in control cultures, with and without thedrug) must be performed to confirm that the anti-fungal agent does not inhibit the growth of the hematopoeitic CFC of interest.

Why does the term ‘over-plated’ mean?• This means that there are too many colonies in the culture and CFC numbers cannot be accurately determined for several reasons.

It is very difficult to accurately identify individual colonies in these cultures, and depletion of essential nutrients or growth factorsand an increase in metabolic by-products may inhibit proliferation. To prevent ‘over-plating’, decrease the number of cells plated inthe assay or plate at 2-3 different cell concentrations to determine optimal input cell density ranges.

Why does the MethoCult® appear to be ‘runny’ and the colonies are floating or smearing?• Thawed MethoCult® medium was not thoroughly mixed before dispensing into tubes.• Incorrect volumes of components or cells were added to the MethoCult®.• Tubes containing cells and the MethoCult® medium were not thoroughly mixed before plating.

Why should MethoCult® be dispensed using blunt-end needles?• Methylcellulose-based media is a viscous solution and cannot be accurately dispensed using pipettes due to the adherence of the

medium to the inside of the pipette. In addition, the use of blunt-end needles prevents injury due to needle pricks.

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6.4 Enumeration of Mouse CFC Assays

• CFC numbers should be evaluated following the recommended incubation period. If the cultures cannot be counted at that time, thecultures can be stored at 33°C, 5% CO2 and ≥95% humidity.

• It is important to use a high quality inverted microscope equipped with low (2.5X) and higher power (4-5X, 10X) objectives. Firstscan on low power to determine the overall density of colonies within the culture. Total colonies and the different CFC classes canthen be determined using 40-50X magnification (10X ocular eyepiece and 4-5X objective). Confirm colony type at highermagnifications as required.

• For specialized applications, such as preparation of cytospins for cytotochemical staining or RNA isolation, it is often necessary toisolate individual colonies from cultures at an earlier time point to ensure a higher proportion of viable cells within the colony. Forexample, individual BFU-E, CFU-GM and CFU-GEMM or the cells from the entire culture can be isolated following 7-10 days ofincubation.







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ProductVolume

CatalogNumber

Components

MC FBS BSA Insulin Transferrin Cytokines

M3434*100 mL

24 x 3 mL0343403444

1% 15% 1% 10 µg/mL 200 µg/mL50 ng/mL rm SCF10 ng/mL rm IL-310 ng/mL rh IL-63 U/mL rh EPO

M3534*100 mL 03534 1% 15% 1% 10 µg/mL 200 µg/mL

50 ng/mL rm SCF10 ng/mL rm IL-310 ng/mL rh IL-6

M3630*100 mL 03630 1% 30% - - - 10 ng/mL rh IL-7

M3334*90 mL 03334 1%‡ 15% 1% 10 µg/mL 200 µg/mL 3 U/mL rh EPO

M3234*80 mL 03234 1%‡ 15% 1% 10 µg/mL 200 µg/mL -

M3231*80 mL 03231 1%‡ 30% 1% - - -

M3236*80 mL 03236 1%‡ - 1% 10 µg/mL 200 µg/mL -

M313440 mL 03134 2.5% - - - - -

7.0 Appendices

7.1 Table 8. MethoCult® Formulations

* These products also contain 10-4 M 2-Mercaptoethanol and 2 mM L-glutamine when diluted as recommended.‡ Final concentration obtained when the contents are brought up to a volume of 100 mL with Iscove’s MDM.

7.2 Preparation of Methylcellulose Medium with PWM-SCCM for BFU-E, CFU-GM andCFU-GEMM Assay

1. Dilute BM cells to 3.0 x 105/mL in Iscove's MDM/2% FBS.2. Set-up cultures as described in Section 3.2. Resulting cultures are 3 x 104 cells per 1.1 mL.3. Incubate for 12-14 days at 37°C, 5% CO2 and ≥95% humidity.4. Identify BFU-E, CFU-GM and CFU-GEMM as described in Section 5.0.

The types and concentrations of cytokines present in PWM-SCCM has not been determined. The total CFC numbers detected in03434 is ~1.5X higher than detected in the presence of 2-5% PWM-SCCM and 3 U/mL rhEpo. For most applications the use ofMethoCult® M3434 (Catalog #03434) is recommended.

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*MethoCult® M3231 (Catalog #03231) can also be used in place of MethoCult® M3134 (Catalog #03134). MethoCult® M3231 contains fetal bovine serum, bovine serum albumin,L-glutamine and 2-Mercaptoethanol, so addition of these components is not necessary.







Table 9: Preparation of PWM-SCCM Containing MethoCult®

Component Catalog Number Volume for 100 mL Final Concentration

Methylcellulose Medium 03134* 40 mL 1%

Fetal Bovine Serum 06200 or 06250 30 mL 30%

10% Bovine Serum Albumin 09300 10 mL 1%

L-glutamine 07100 1 mL 2 mM

PWM-SCCM 02100 2-5 mL 2-5%

rh Erythropoietin 02625 3 U/mL

2-Mercaptoethanol - 0.1 mL of 10-1 M 10-4 M

Iscove’s MDM 36150 bring to 100 mL

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7.3 Cloning of Cell Lines in Methylcellulose-Based Medium

Methylcellulose-based medium can be used for the cloning of non-adherent cell lines from different tissues and from different species. It canalso be used for the quantitation and selection of clones of genetically modified primary hematopoietic cells or cell lines.

Which MethoCult® medium should I use?• For mouse myeloid hematopoietic cell lines, MethoCult® products such as Catalog #03234 or 03231 are suitable. These products

are supplied at 80 mL per bottle allowing researchers to add additional components. ClonaCell®-TCS (Catalog #03821, 03822,03823) is a kit specially formulated for the cloning of transfected non-adherent cells. Please refer to our website(www.stemcell.com) for more information.

Can I use MethoCult® medium if my cell line is not usually cultured in Iscove's MDM?• Many cell lines will grow in MethoCult® medium to which other cell culture media such as Dulbecco's Modified Eagle's Medium

(DMEM), Minimum Essential Medium (MEM), or RPMI 1640 is added. Culture media, sera and other components are added toMethoCult® base medium to achieve a final volume of 100 mL. Contact us for information and pricing on custom methylcellulose-based media.

For cloning, how many cells should be cultured per 35 mm dish?• The optimal number of cells to place in the MethoCult® medium will be dependent on the cell lines and application. To allow easy

isolation of individual clones and prevent depletion of required cytokines or medium nutrients, the number of colonies should notexceed ~150 per 35 mm dish. For example, if the expected cloning efficiency is 50%, then cells should be plated at 200-300 per35 mm dish.

How are selective drugs added to MethoCult® medium?• Drugs or other compounds should be added directly to MethoCult® and well mixed prior to the addition of cells. It is important to

maintain the correct viscosity of the methylcellulose-based medium. To add drugs or other compounds to complete formulationsadd appropriate number of cells in a smaller volume (Example: To 3 mL tube, add drug in 100 µL and cells in 200 µL). To useformulations such as Catalog #03234 or 03231, dispense 2.4 mL methylcellulose medium per tube, add desired supplements,growth factors and drug to a final volume of 3.0 mL and mix well, prior to addition of cells.

ProcedureGeneral Procedure for Cloning Cell Lines in Methylcellulose-Based Media

1. Thaw MethoCult® base medium overnight under refrigeration (2-8°C) or at room temperature.2. Add desired components such as fetal bovine serum (FBS), supplements, growth factors and culture medium to achieve a final

volume of 100 mL.3. Mix components well by shaking bottle vigorously for 1-2 minutes. Let stand for 3-5 minutes to allow bubbles to rise.4. Dispense medium into sterile culture tubes using a syringe and 16 gauge blunt-end needle (Catalog #28110, 28210), as described in

Section 3.1.5. Add cells to methylcellulose-based medium at 1:10 ratio (0.3 mL cells: 3 mL methylcellulose for duplicate cultures; 0.4 mL cells: 4 mL

methylcellulose for triplicate cultures; 1.5 mL cells: 15 mL methylcellulose for 12-13 replicate cultures; etc.).6. Vortex tube to ensure all cells and components are thoroughly mixed. Let stand for 5 minutes to allow bubbles to dissipate. Dispense

1.1 mL into each 35 mm dish with lid using 3 cc syringe and 16 gauge blunt-end needle.7. Place dishes in 100 mm petri dish or square culture dish with one or more open 35 mm dishes containing 3-4 mL sterile water.8. Culture at 37°C in a humidified incubator with 5% CO2 in air for 7-14 days until colonies become visible using inverted microscope or

are macroscopic in size.9. Individual colonies can be "picked" using a pipettor and 200 µL sterile pipette tips or a glass Pasteur pipette with elongated tip in a

volume of 25-50 µL and diluted into suitable culture medium in 96- or 24-well culture plates for expansion of cell numbers.

methyl cellulose method

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