Measurement of Human and Murine Stem Cell Factor (c‐kit Ligand)

Kent A. Smith1, Krisztina M. Zsebo2

1 Amgen, Thousand Oaks, California, 2 Cell Genesys, Foster City, California
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 6.17
DOI:  10.1002/0471142735.im0617s04
Online Posting Date:  May, 2001
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Stem cell factor (SCF) is an early‐acting, hematopoietic growth factor that binds to the receptor encoded by the proto‐oncogene c‐kit. It is a potent growth factor for primitive bone marrow cells as well as thymocytes. This unit describes three protocols for detecting human and murine SCF. In the first, human or rodent SCF is measured by its ability to stimulate proliferation of the human megakaryoblastic leukemia cell line, UT‐7. Because rat and mouse SCF bind well to human c‐kit, human and rodent SCF can both be measured using the first basic protocol. In an , rodent SCF is assayed by its ability to stimulate proliferation of the clonal murine mast cell line, MC/9. Human SCF is not very active on rodent cells and thus cannot be measured using this protocol. Both of the cell proliferation assays lack specificity because they are capable of detecting other cytokines in addition to SCF. The third protocol is a radioreceptor assay using the human erythroleukemia cell line, OCIM1; it specifically measures murine or human SCF and not other cytokines. Support protocols describe maintenance of UT‐7 and MC/9 cells and preparation of plasma membranes from OCIM1 cells.

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Table of Contents

  • Basic Protocol 1: UT‐7 Cell Proliferation Assay for Murine and Human SCF Activity
  • Support Protocol 1: Maintenance of UT‐7 Cells
  • Alternate Protocol 1: MC/9 Cell Proliferation Assay for Murine SCF Activity
  • Support Protocol 2: MC/9 Cell Line Maintenance
  • Basic Protocol 2: Radioreceptor Assay for Murine and Human SCF
  • Support Protocol 3: Maintenance of OCIM1 Cells and Isolation of Plasma Membranes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: UT‐7 Cell Proliferation Assay for Murine and Human SCF Activity

  • recipeHuman or rodent recombinant SCF standard (rSCF; see reagents and solutions)
  • Unknown samples containing SCF
  • Complete RPMI‐4 ( appendix 2A) without 2‐ME
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 5 × 104 UT‐7 cells/ml in complete RPMI‐4 (first protocol 2support protocol)
  • 96‐well polystyrene microtiter plates (Falcon #3072)
  • Additional reagents and equipment for labeling cells and determining [3H]thymidine incorporation ( appendix 3A)

Support Protocol 1: Maintenance of UT‐7 Cells

  • UT‐7 cell line (Miura et al., )UT‐7 growth medium: complete IMDM‐10 ( appendix 2A) without 2‐ME, containing 5 ng/ml human recombinant granulocyte/macrophage colony stimulating factor (hrGM‐CSF; Table 97.80.4711)
  • Phosphate‐buffered saline (PBS; appendix 2A), sterile
  • Complete RPMI‐4 ( appendix 2A), without 2‐ME
  • 175‐cm2 tissue culture flasks
  • Additional reagents and equipment for counting viable cells ( appendix 3A)

Alternate Protocol 1: MC/9 Cell Proliferation Assay for Murine SCF Activity

  • MC/9 cell line (ATCC #CRL 8306)
  • MC/9 growth medium: complete DMEM‐10 ( appendix 2A) containing 20% (v/v) concanavalin A–activated supernatant (Con A–sup; unit 3.13) prepared using rat spleen cells
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • Complete DMEM‐10 ( appendix 2A)
  • Complete RPMI‐4 ( appendix 2A)
  • 175‐cm2 tissue culture flasks
  • Additional reagents and equipment for counting viable cells ( appendix 3A)

Support Protocol 2: MC/9 Cell Line Maintenance

  • recipeHuman or rodent recombinant SCF standard (rSCF; see reagents and solutions)
  • Assay buffer: 0.25% (w/v) bovine serum albumin (BSA) in 50 mM Tris⋅Cl, pH 7.5 ( appendix 2A), ice‐cold (store ≤1 month at 4°C)
  • Unknown samples containing SCF
  • 58.0 mCi/mg (1 mCi/ml) human [125I]SCF (e.g., ICN Radiochemicals)
  • OCIM1 plasma membrane preparation diluted in assay buffer to yield 20% specific binding
  • Lead foil
  • Beckman J2‐HS centrifuge with JA‐18.1 rotor (or equivalent)
  • 21‐G needle and syringe, attached to vacuum flask with rubber tubingγ‐scintillation counter capable of quantifying microcentrifuge tubes (Pharmacia LKB Nuclear)
NOTE: Perform all steps of this protocol on ice.

Basic Protocol 2: Radioreceptor Assay for Murine and Human SCF

  • OCIM1: human erythroleukemic cell line (available from T. Papayannopoulou, University of Washington, Seattle)
  • Complete IMDM‐10 ( appendix 2A)
  • Phenylmethylsulfonyl fluoride (PMSF; Sigma)
  • Phosphate‐buffered saline (PBS) without Ca++ and Mg++ ( Irvine Scientific)
  • Phosphate‐buffered saline (PBS) with 1000 mg/ml D‐glucose and 36 mg/liter sodium pyruvate (GIBCO/BRL), ice‐cold
  • Sucrose buffer: 0.25% (w/v) sucrose in TE buffer, ice‐cold (sterilize using 0.45‐µm fiter; store ≤1 yr, 4°C)
  • 36% (w/v) sucrose in H 2O, ice cold (store ≤1 yr at 4°C)
  • TE buffer, pH 7.0 ( appendix 2A), ice‐cold
  • 175‐cm2 tissue culture flasks
  • 500‐ml polystyrene centrifuge tubes
  • Beckman J2‐HS centrifuge with JA‐10, JA‐18.1, and JA‐20 rotors (or equivalent)
  • 50‐ml centrifuge tubes
  • Sorvall RT‐6000B centrifuge (or equivalent)
  • 45‐ml cell‐disruption bomb ( Parr Instrument), 4°C
  • Hemacytometer
  • 40‐ml screwcap centrifuge tubes (Nalgene)
  • 38.5‐ml polyallomer ultracentrifuge tubes (e.g., Beckman #326823)
  • 20‐ml syringe and 18‐G, 11/2‐in. needle (Becton Dickinson)
  • Beckman L‐70 ultracentrifuge with SW‐28 rotor (or equivalent)
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Literature Cited

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