CBSC were cultured with rNK cells or aNK cells or with CXCL9 (Peprotech) at different concentrations for 4 hrs. A minimum of 200 CBSC were plated in MethoCult GF H-84434 (Stemcell Technologies) and cultured for 14 days at 37°C, 5% CO2. Colony formation was enumerated using an inverted microscope at the end of culture. For integrin blocking experiments, CBSC were incubated with blocking antibodies (eBioscences) against β7 integrin (FIB504), CD11a (HI111), CD49d (9F10) and CD49e (IIA1) at 10 μg/mL for 1 h and then washed before co-culture with NK cells and CFU assays.
Methocult gf h84434
Manufactured by STEMCELL
Sourced in Canada
MethoCult GF H84434 is a semi-solid culture medium designed for the growth and enumeration of human hematopoietic progenitor cells. The medium contains recombinant human growth factors and cytokines that support the growth of different progenitor cell types.
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5 protocols using methocult gf h84434
1
Colony formation of CBSC in NK cell co-culture
2
Hematopoietic Stem Cell Assay
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At 3–5 d after transduction, 4,000–5,000 GFP+ CD34+ cells were sorted and plated into a 6-well plate with methylcellulose-containing medium (MethoCult GF H84434 ; StemCell Technologies). After 14 d, the number of colonies was analyzed, and 5 × 104 CD45+ cells were replated into a second methylcellulose assay. Colony types were determined after the first plating round. To determine the colony formation of 1° recipient mice, 2.5 × 104 – 1.25 × 105 hCD45+ GFP+ BM cells were sorted, and colony assays were performed. Duplicate or triplicate wells were set up for each condition.
3
Colony Forming Unit Assay
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On day 3 of coculture, 2000 mononuclear cells (MNCs) from HS-5-Veh (PBS) condition and in HS-5-TNFα condition were resuspended in IMDM (Thermo Fisher Scientific). This cell suspension was mixed with MethoCult GF H84434 (STEMCELL Technologies), which allows the growth of colonies from all three lineages, and triplicate dishes were plated. The MethoCult plates were kept at 37°C in a 5% CO2 incubator for 2 weeks until colony counting under a light microscope (Zeiss).
4
Colony Formation Assay for Hematopoietic Cells
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Washed labeled or mock-labeled cells were resuspended in methylcellulose containing medium (Methocult GF H84434, Stemcell Techonologies, Vancouver, BC, Canada) and seeded in triplicate at 500 cells per 35 mm dish. Dishes were incubated for 14 days in a humidified tray at 37°C and 5.0% CO2, after which two trained non-blinded observers enumerated the colonies. We accepted an interobserver variation of 10%. Three types of colonies were distinguished: burst forming unit-erythroid (BFU-E), colony forming unit-granulocyte/monocyte (CFU-GM) and colony forming unit-granulocyte/erythrocyte/monocyte/megakaryocyte (CFU-GEMM).
5
Bone Marrow Toxicity of Venadaparib
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The experiment for bone marrow toxicity of venadaparib was performed at Stemcell Technologies. Bone marrow–derived hematopoietic progenitor cells from three lots of human bone marrow mononuclear cells were incubated with venadaparib (0.000169–30 μmol/L) or a control compound, 5-fluorouracil (F6627; Sigma-Aldrich), in MethoCult GF H84434 (catalog no. 84434; Stemcell Technologies) at 37°C in a 5% CO2 incubator. After 14 days, the hematopoietic colonies were assessed and scored by trained personnel. The mean colony number was calculated for triplicate cultures under each condition and normalized to the solvent controls. Standard t tests were performed to compare the solvent controls for each test compound. Because of the potential subjectivity of colony enumeration, a P value of less than 0.01 was considered significant.
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