Methyl Cellulose Method

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<p>Mouse Colony-Forming Cell Assays Using MethoCultTECHNICAL MANUALCatalog #28405</p> <p>StemCell TechnologiesThe Cell Experts</p> <p>i</p> <p>FOR RESEARCH USE ONLYIn North America Tel: 604.877.0713 Fax: 604.877.0704 Toll Free Tel: 1.800.667.0322 Toll Free Fax: 1.800.567.2899 e-mail: info@stemcell.com e-mail: orders@stemcell.com In the United Kingdom Tel: +44.(0).20.7537.7565 Fax: +44.(0).20.7515.5408 Toll Free within United Kingdom: Tel: 0800.731.27.14 Fax: 0800.731.27.13 e-mail: info@stemcellgb.com In Europe Tel: +33.(0).4.76.04.75.30 Fax: +33.(0).4.76.18.99.63 e-mail: info@stemcellfrance.com</p> <p>StemCell TechnologiesVersion 3.1.1 June 2005 Catalog # 28405</p> <p>ii</p> <p>Table of Contents1.0 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2.0 StemCell Technologies Products for Mouse CFC Assays . . . . . . . . . . . . . . . . . . . . . . . . . 1 3.0 Storage and Handling of MethoCult Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33.1 Thawing and Dispensing of Complete MethoCult Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3.2 Thawing and Dispensing of Incomplete MethoCult Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4</p> <p>4.0 Mouse Hematopoietic CFC Assays in MethoCult Media . . . . . . . . . . . . . . . . . . . . . . . . . 54.1 Procedure Diagram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Equipment and Reagents Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3 Mouse Cell Isolation Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.1 Bone Marrow Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.2 Spleen Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.3 Peripheral Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3.4 Fetal Liver, Day 10-16 PC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4 Cell Counts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5 General Method for Set-up of CFC Assays in MethoCult Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.6 Mouse CFU-E Assay and Mature BFU-E Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.7 Mouse BFU-E, CFU-GM, CFU-GEMM Assay, and CFU-GM Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.8 Mouse CFU Pre-B Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 6 6 6 7 7 7 8 9 10 10 11</p> <p>5.0 Descriptions and Photographs of Mouse Hematopoietic CFCs . . . . . . . . . . . . . . . . . . . . . 12 6.0 Frequently Asked Questions and Helpful Hints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156.1 6.2 6.3 6.4 MethoCult Media and Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preparation of Cell Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Set-up and Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enumeration of Mouse CFC Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 15 15 17</p> <p>7.0 Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187.1 MethoCult Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 7.2 Preparation of Methylcellulose Medium with PWM-SCCM for BFU-E, CFU-GM and CFU-GEMM assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 7.3 Cloning of Cell Lines in Methylcellulose-Based Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20</p> <p>FOR RESEARCH USE ONLYIn North America Tel: 604.877.0713 Fax: 604.877.0704 Toll Free Tel: 1.800.667.0322 Toll Free Fax: 1.800.567.2899 e-mail: info@stemcell.com e-mail: orders@stemcell.com In the United Kingdom Tel: +44.(0).20.7537.7565 Fax: +44.(0).20.7515.5408 Toll Free within United Kingdom: Tel: 0800.731.27.14 Fax: 0800.731.27.13 e-mail: info@stemcellgb.com In Europe Tel: +33.(0).4.76.04.75.30 Fax: +33.(0).4.76.18.99.63 e-mail: info@stemcellfrance.com</p> <p>StemCell TechnologiesVersion 3.1.1 June 2005 Catalog # 28405</p> <p>iii</p> <p>FOR RESEARCH USE ONLYIn North America Tel: 604.877.0713 Fax: 604.877.0704 Toll Free Tel: 1.800.667.0322 Toll Free Fax: 1.800.567.2899 e-mail: info@stemcell.com e-mail: orders@stemcell.com In the United Kingdom Tel: +44.(0).20.7537.7565 Fax: +44.(0).20.7515.5408 Toll Free within United Kingdom: Tel: 0800.731.27.14 Fax: 0800.731.27.13 e-mail: info@stemcellgb.com In Europe Tel: +33.(0).4.76.04.75.30 Fax: +33.(0).4.76.18.99.63 e-mail: info@stemcellfrance.com</p> <p>StemCell TechnologiesVersion 3.1.1 June 2005 Catalog # 28405</p> <p>1</p> <p>1.0 IntroductionIn the adult mouse bone marrow (BM), a small number of hematopoietic stem cells (HSCs) produce heterogeneous populations of actively dividing hematopoetic progenitors. These hematopoietic progenitors proliferate and differentiate resulting in the generation of millions of mature blood cells daily. In vitro assay systems have been developed to quantify multi-potential progenitors and lineage-restricted progenitors of the erythroid, granulocytic, monocyte-macrophage, and megakaryocyte-myelopoietic pathways as well as a subset of mouse pre-B lymphoid cells. When cultured in a suitable semi-solid matrix, individual progenitors called colony-forming cells (CFCs), proliferate to form discrete 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 37C for periods ranging from a few days to several weeks. The CFCs are classified and enumerated based on the morphological recognition of one or more types of hematopoietic lineage cells within the colony. This can 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 a relatively 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 culture medium 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 permits better growth of erythroid lineage cells than other types of semi-solid matrices, thus allowing the assay of erythroid, granulocyte, monocyte and multi-potential CFCs within the same culture.</p> <p>2.0 StemCell Technologies Products for Mouse CFC AssaysMethoCult Methylcellulose-Based Medium StemCell Technologies (StemCell) rigorously screens and selects components used in the manufacture of MethoCult products. It is known that different batches of methylcellulose, fetal bovine serum (FBS) and bovine serum albumin (BSA) vary widely in their ability to promote CFC growth. If using media components other than those pre-screened and available from StemCell, it is important to test components individually and 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 normal mouse 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 lack components 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 StemCell Technologies for more information.</p> <p>FOR RESEARCH USE ONLYIn North America Tel: 604.877.0713 Fax: 604.877.0704 Toll Free Tel: 1.800.667.0322 Toll Free Fax: 1.800.567.2899 e-mail: info@stemcell.com e-mail: orders@stemcell.com In the United Kingdom Tel: +44.(0).20.7537.7565 Fax: +44.(0).20.7515.5408 Toll Free within United Kingdom: Tel: 0800.731.27.14 Fax: 0800.731.27.13 e-mail: info@stemcellgb.com In Europe Tel: +33.(0).4.76.04.75.30 Fax: +33.(0).4.76.18.99.63 e-mail: info@stemcellfrance.com</p> <p>StemCell TechnologiesVersion 3.1.1 June 2005 Catalog # 28405</p> <p>2Table 1: MethoCult Products and Applications Catalog #03434 03444 03534 03630 03334 03234</p> <p>Containsrm SCF, rm IL-3, rh IL-6, rh Epo rm SCF, rm IL-3, rh IL-6 rh IL-7 rh Epo Without cytokines</p> <p>ApplicationsDetection of BFU-E, CFU-GM, CFU-GEMM in BM, spleen, PB and fetal liver Detection of CFU-GM in BM, spleen, PB and fetal liver Detection of CFU-pre-B in BM Detection of CFU-E, mature BFU-E 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 Allows researchers to add cytokines of their choice in defined serum-free methylcellulose-based medium Preparation of methylcellulose-based medium for specialized applications Detection of CFU-Mk in BM and other hematopoietic tissues. See our website or contact your StemCell Technical Representative.</p> <p>03231</p> <p>Without cytokines</p> <p>03236 03134 MegaCult-C</p> <p>Serum-free without cytokines Methylcellulose base Collagen-based medium without cytokines</p> <p>BM: Bone Marrow; PB: Peripheral Blood</p> <p>Table 2: Recombinant Cytokines for Culture of Mouse Hematopoietic Cells Catalog #02715, 02915 02732, 02932 02733, 02903 02751, 02951 02731, 02931 02720, 02920 02620, 02820 02625 02100 Additional Cytokines</p> <p>Descriptionrm G-CSF. For growth of granulocytic progenitors rm GM-CSF. For growth of granulocytic and monocytic progenitors rm IL-3. Used in combination with other cytokines to promote growth of early myeloid progenitors of all lineages rm M-CSF. For growth of monocytic progenitors rm SCF. For growth of mast cells and used in combination with other cytokines to promote growth of myeloid and lymphoid progenitors rm TPO or rh TPO. Used in combination with other cytokines to promote growth of megakaryocytic progenitors rh EPO. Used in combination with other cytokines for growth of erythroid progenitors PWM-SCCM: Pokeweed Mitogen Spleen Cell Conditioned Medium. Used as source of colony stimulating factors Refer to StemCell Technologies Catalog, website or contact your Technical Representative for a complete list of available cytokines.</p> <p>FOR RESEARCH USE ONLYIn North America Tel: 604.877.0713 Fax: 604.877.0704 Toll Free Tel: 1.800.667.0322 Toll Free Fax: 1.800.567.2899 e-mail: info@stemcell.com e-mail: orders@stemcell.com In the United Kingdom Tel: +44.(0).20.7537.7565 Fax: +44.(0).20.7515.5408 Toll Free within United Kingdom: Tel: 0800.731.27.14 Fax: 0800.731.27.13 e-mail: info@stemcellgb.com In Europe Tel: +33.(0).4.76.04.75.30 Fax: +33.(0).4.76.18.99.63 e-mail: info@stemcellfrance.com</p> <p>StemCell TechnologiesVersion 3.1.1 June 2005 Catalog # 28405</p> <p>3Table 3: Support Products for Mouse Cells Catalog #06200, 06250 07050 07060 07700 07800, 07850 09300 09500 09600, 09650 27100, 27150 27305 27500 28110, 28120</p> <p>DescriptionFetal Bovine Serum. Prescreened and selected for growth of mouse hematopoietic cells in suspension or methylcellulose-based cultures. Trypan Blue. For cell viability counts 3% Acetic Acid with Methylene Blue. For nucleated cell counts Iscove's MDM with 2% FBS. For cell washing Ammonium Chloride. For lysis of red blood cells Detoxified 10% BSA Solution (in Iscoves MDM) BIT 9500 (5X media supplement containing BSA, transferrin and insulin). Prescreened and selected for growth of mouse hematopoietic cells in suspension or methylcellulose-based cultures. StemSpan SFEM. For growth of hematopoietic cells in suspension cultures. Pre-tested 35 mm culture dishes Cell strainer, 40 m. For peparation of single cell suspensions Gridded Scoring Dishes Blunt-End Needles. For dispensing of methylcellulose-based medium.</p> <p>3.0 Storage and Handling of MethoCult MediaMethoCult media should be stored at -20C. Store for up to one month at 2-8C. Repeated freezing and thawing is not recommended.</p> <p>3.1 Thawing and Dispensing of Complete MethoCult MediaComplete MethoCult media (Catalog #03434, 03534, 03630) is supplied at 100 mL per bottle. It is formulated to allow the addition of cells to the 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 be accurately 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.</p> <p>FOR RESEARCH USE ONLYIn North America Tel: 604.877.0713 Fax: 604.877.0704 Toll Free Tel: 1.800.667.0322 Toll Free Fax: 1.800.567.2899 e-mail: info@stemcell.com e-mail: orders@stemcell.com In the United Kingdom Tel: +44.(0).20.7537.7565 Fax: +44.(0).20.7515.5408 To...</p>

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