Hematopoietic cells collected from day +12 of differentiation cultures were tested on clonogenic assays with and without TKIs. For each condition, 1 × 104 cells were plated in methylcellulose (MethoCultTM H4434, STEMCELL Technologies) with a TKI drug (alone or in combination) at the following concentrations: 1 μM Imatinib (Sigma-Aldrich, SML1027) and 1 μM Ruxolitinib (Selleckchem, Planneg, Germany, S1378). The number of clonogenic growth was evaluated at day +14. All experiments were performed in triplicates.
Methocult h4434
Manufactured by STEMCELL
Sourced in Canada, France, United States
MethoCult H4434 is a semisolid methylcellulose-based medium designed for the colony-forming unit (CFU) assay of human hematopoietic progenitor cells. It supports the growth and differentiation of granulocyte, erythroid, macrophage, and megakaryocyte progenitor cells from human bone marrow, peripheral blood, and cord blood samples.
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Lab products found in correlation
Methocult m3434, by STEMCELL (14 mentions)
Penicillin streptomycin, by Merck Group (4 mentions)
Myelocult h5100, by STEMCELL (3 mentions)
Methocult h4100, by STEMCELL (3 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (3 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (3 mentions)
Stem cell factor (scf), by STEMCELL (3 mentions)
Interleukin 3 (il 3), by STEMCELL (3 mentions)
Gm csf, by STEMCELL (3 mentions)
Methocult gf m3434, by STEMCELL (2 mentions)
Fetal bovine serum (fbs), by STEMCELL (2 mentions)
M3434, by STEMCELL (2 mentions)
P3 primary cell 4d nucleofector x kit, by Lonza (2 mentions)
Dmpge2, by Cayman Chemical (2 mentions)
Flt3l, by STEMCELL (2 mentions)
Stemspan, by STEMCELL (2 mentions)
Facsaria, by BD (2 mentions)
Bit serum replacement, by Thermo Fisher Scientific (2 mentions)
Myelocult m5300, by STEMCELL (2 mentions)
4 oht tamoxifen, by Merck Group (2 mentions)
107 protocols using methocult h4434
1
Clonogenic Assay of Hematopoietic Cells with TKIs
2
Pesticide-Exposed BM-MSCs and CD34+ Hematopoietic Progenitor Assay
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BM-MSCs were exposed to pesticides only during the 21 days of expansion culture before co-cultures with normal CD34+ cells, and CAFC assays were performed as previously described and as detailed in supplementary methods [22 (link)]. Briefly, CD34+ cells were thus co-cultured with the pesticide-exposed BM-MSCs for 7 days (37 °C, 5% CO2), seeded at 6000 CD34+/cm2 (ratio CD34+ cells/BM-MSCs = 1/3) in Myelocult™ H5100 medium (StemCell Technologies, Vancouver, Canada). At the end of coculture, CD34+ cells were sorted (BD FACSMelodyTM cell sorter, Becton-Dickinson) and cultured on MS5 stromal cells [25 (link)] for 5 additional weeks in 96-well plates (37 °C, 5% CO2) at 6 different dilutions (range 1–100 CD34+ cells/well) in 0.2 mL of Myelocult™ H5100 medium. After 5 weeks of co-culture, each well was examined by inverted phase contrast microscopy (DMI 3000 B, Leica) to identify cobblestone areas. The percentage of wells with at least one CAFC was quantified and the frequency of CAFC in the initial CD34+ cell population was calculated as previously described [26 (link)]. The CAFC proliferative capacity was determined by subculturing the wells containing one cobblestone area in semi-solid medium (MethoCultTM H4434, StemCell Technologies) to determine the mean total number of clonogenic progenitors (including CFU-GM, BFU-E and CFU-M) generated by each CAFC.
3
Long-Term Culture-Initiating Cell Assay
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LTC-IC assays have been performed according to previously reported techniques [19 (link)]. Briefly, cells collected at day +12 of hematopoietic differentiation were counted and started on long-term culture assays in triplicates using 4.5 × 104 cells/dish. Cultures were maintained at 33 °C on MS-5 stromal cells with weekly half-medium changes (MyeloCultTM H5100, STEMCELL Technologies). At week +5, non-adherent and adherent cells were collected, counted, and plated in methylcellulose (MethoCultTM H4434, STEMCELL Technologies, Grenoble, France) at the concentration of 5 × 103 cells/dish in triplicates. The number of clonogenic growth was evaluated at days +14 and +21.
4
Colony Forming Cell Evaluation
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The evaluations of colony forming cells (CFC) were conducted after three days in co-culture with endothelial cells. Briefly, 3 × 103 hematopoietic cells from different co-cultures, were sub-cultured for 14 days in complete methylcellulose medium with recombinant cytokines (MethoCultTM H4434, StemCell Technologies Inc., Vancouver, BC, Canada). CFC were defined as clusters consisting of 40 or more cells and were quantified using an inverted microscope (CKX41SF, Olympus Corporation, Tokyo Japan) under 4X and 10X. The counting of CFC was normalized with the initial number of CFC at time zero and after enrichment, of each sample.
5
Hematopoietic Differentiation: Colony Assay
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Non-adherent cells collected at day +12 of hematopoietic differentiation were counted and plated in methylcellulose-based medium (MethoCultTM H4434, STEMCELL Technologies, Grenoble, France) at the concentration of cells/mL and incubated for 14 days in a 37 °C incubator with 5% CO2. On day +14, colonies were enumerated.
6
Colony Forming Assay for Mouse and Human HSPCs
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For mouse HSPCs, total cells derived from 500 edited LSK cells were added to 3 mL of MethoCult M3434 (STEMCELL Technologies) and plated in triplicate. For human HSPCs, total cells derived from 500 edited CD34+CD38−CD45RA−CD90+ cells were added to 4 mL of MethoCult H4434 (STEMCELL Technologies) and plated in triplicate. Plates were incubated at 37°C in 5% CO2. The number of colonies in each sample was scored at 7 days (for mouse HSPCs) or 14 days (for human HSPCs).
7
Clonogenic Capacity of Leukemic Progenitors
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The clonogenic capacity of leukemic progenitors was evaluated by colony-forming unit (CFU) assays. Briefly, AML cells (500–25000 cells/well) were seeded in semisolid methylcellulose media (Methocult #H4434; Stemcell Technologies) according to established protocols. Progenitor assays of xenografted leukemic cells were performed following human cell purification as described above. Individual CFU wells were seeded from multiple mice.
8
Hematopoietic Colony Quantification
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Clonogenic assays were performed using methylcellulose-based medium (MethoCult H4434, STEMCELL Technologies). Fourteen days after seeding of the cells, hematopoietic colonies (total colonies and CFU-GM and BFU-E colonies) were scored under an inverted phase-contrast microscope at ×40 (Nikon Diaphot). An appropriate number of total or purified BM cells were seeded to facilitate the scoring of 10–100 colonies per plate.
9
Clonogenic Cytotoxicity Assay for AML Cell Lines and Primary Cells
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To evaluate the toxicity of the bioconjugate on clonogenic progenitor cells, AML cell lines as well primary human cells were incubated with the compound for 4 hours. Subsequently, cells were washed and cell counts were determined via trypan blue exclusion. Colony forming cell unit (CFC) assays were performed as described previously14 (link). In all cases cell numbers placed in methylcellulose were calculated according to the initial cell number at the start of the experiment, not taking into account any cell loss during the incubation time. 1000 cells (OCI-AML3 and HL60) and 500 cells (THP1) were plated per dish (methocult H4330). For primary patient samples, 10,000 cells were plated per dish in methylcellulose H4330 (Stem Cell Technologies, Vancouver, BC, Canada) and supplemented with cytokines. For the CD34+ enriched CB cells, 300 cells were plated per dish in methocult H4434 (Stem Cell Technologies, Vancouver, BC, Canada). Each experiment was performed in duplicates and values mentioned are Mean ± SEM.
10
Clonogenic Potential of Leukemia Cells
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As reported previously [11 ], clonogenic potential was assessed through colony growth in the presence of dimethylsulfoxide (vehicle), JDM‐7 or tadalafil at concentrations as indicated. Human MOLM‐13, MV4‐11 and THP‐1 cells were plated in methylcellulose media (Methocult H4100; Stemcell Technologies, Cologne, Germany) supplemented with 10% fetal bovine serum in triplicates at a cell dose of 1000 per plate for MOLM‐13 and 2000 for MV4‐11 and THP‐1. Cord blood cells were plated in methylcellulose media (Methocult H4434; Stemcell Technologies) at a cell dose of 25 000 per plate. Cells were incubated at 37 °C in 5% CO2 for the indicated number of days, after which time colonies were counted. The variance between the groups is statistically similar. Three replicated experiments were performed. P < 0.05 was considered statistically significant.
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