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Mouse Colony-Forming Cell Assays Using MethoCultTECHNICAL MANUALCatalog #28405

StemCell TechnologiesThe Cell Experts

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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

StemCell TechnologiesVersion 3.1.1 June 2005 Catalog # 28405

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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

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

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

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

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

StemCell TechnologiesVersion 3.1.1 June 2005 Catalog # 28405

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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

StemCell TechnologiesVersion 3.1.1 June 2005 Catalog # 28405

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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.

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.

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

StemCell TechnologiesVersion 3.1.1 June 2005 Catalog # 28405

2Table 1: MethoCult Products and Applications Catalog #03434 03444 03534 03630 03334 03234

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

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

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