Colony Forming Unit (CFU) formation from cloned wtCSCs vs. WCreCSCs was assessed by plating relative cells in 1.2 ml of MethoCult medium (STEMCELL Technologies, Vancouver, Canada) (1% methylcellulose in CSC growth medium). Colonies were counted at 14 days after plating. The n-value for the biological replicates is specified in the relative figure legends.
Methocult medium
Manufactured by STEMCELL
Sourced in Canada
MethoCult medium is a semisolid culture medium used for the clonal growth and enumeration of hematopoietic progenitor cells from human, mouse, and non-human primate samples. It contains the necessary growth factors and nutrients to support the formation of colonies from various types of progenitor cells.
Automatically generated - may contain errors
Lab products found in correlation
Megacult c medium, by STEMCELL (3 mentions)
Mammocult proliferation supplement, by STEMCELL (2 mentions)
Mammocult basal medium human, by STEMCELL (2 mentions)
Stemvision instrument and software, by STEMCELL (2 mentions)
L calc limiting dilution analysis software, by STEMCELL (2 mentions)
Granulocyte macrophage csf, by STEMCELL (1 mentions)
Interleukin 3 (il 3), by STEMCELL (1 mentions)
Interleukin 6 (il 6), by STEMCELL (1 mentions)
Anti cd45ra, by BioLegend (1 mentions)
Aria cell sorter, by BD (1 mentions)
Anti cd38, by BioLegend (1 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions)
Anti cd15, by BioLegend (1 mentions)
Anti cd34, by BioLegend (1 mentions)
Anti cd90, by BD (1 mentions)
Fetal bovine serum (fbs), by Cambrex (1 mentions)
Ammonium chloride solution, by STEMCELL (1 mentions)
Methocult gf m3434, by STEMCELL (1 mentions)
Heparin, by Merck Group (1 mentions)
M3234, by STEMCELL (1 mentions)
57 protocols using methocult medium
1
CFU Assay for Cloned wtCSCs vs. W^Cre^CSCs
2
Hematopoietic Colony Formation Assay
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Single cells (5 × 103 or 104) were plated into 1.5 ml of Methocult medium containing SCF, granulocyte colony-stimulating factor (CSF), granulocyte macrophage CSF, IL-3, IL-6, and erythropoietin (StemCell Technologies, Inc., Vancouver, BC, Canada, http://www.stemcell.com ) in 35-mm-dish low-attachment plates (Greiner Bio-One), incubated at 37°C in a humid chamber, and scored for hematopoietic colony formation after 12–15 days.
3
Clonogenic Assay for Leukemic Cells
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10,000 BCR-ABL1-transformed ALL cells or 100,000 CML-like cells were used for this assay. Cells were resuspended in murine MethoCult medium (StemCell Technologies, Vancouver, BC, Canada) and plated on dishes (3 cm in diameter) with an extra dish of water to prevent evaporation. After 7 to 14 days, colonies were counted.
4
Assaying Hematopoietic Progenitor Cells
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Assays of colony forming unit megakaryocyte (CFU-MK) and colony forming unit myeloid (CFU-myeloid) cells were performed on mouse lineage negative cells. For CFU-MK assay, 5,000 bone marrow lineage negative cells were seeded in Megacult-C medium (Stemcell Technologies) supplemented with 10ng/ml mouse IL-3, 10 ng/ml human IL-6, and 50 ng/ml of mouse THPO and cultured in the presence of DMSO or chemical inhibitors for 7–9 days. For CFU-myeloid assay, 2,000 bone marrow lineage negative cells were seeded in MethoCult medium (Stemcell Technologies) supplemented with 10ng/ml mouse IL-3, 10 ng/ml human IL-6, 10 ng/ml mouse SCF and 10 ng/ml of mouse granulocyte-monocyte colony stimulating factor (GM-CSF), and cultured in the presence of DMSO or chemical inhibitors for 7 days. Slides of Megacult cultures were fixed with methanol and then were stained with substrates of acetylcholinesterase according to the manufacturer’s instructions. Only stained colonies were counted. For the CFU-myeloid assay, CFU-G, CFU-M and CFU-GM were enumerated on day 7 according to the manufacturer’s instructions. The number of CFU-myeloid colonies was taken as the sum of CFU-G, CFU-M and CFU-GM.
5
Clonal versus Freshly Isolated CSC CFU Assay
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CFU formation from cloned versus freshly isolated CSCs was assessed by plating relative cells in 1.2 ml of MethoCult medium (STEMCELL Technologies, Vancouver, Canada) (1% methylcellulose in CSC growth medium). Colonies were counted at 14 days after plating.
6
Cell Cycle and Clonogenic Assay
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1 × 104 cells were cultured in 24-well plate, then cells were counted at indicated time. After incubation, cells were washed with PBS. The pellets were fixed in 70% ethanol, washed in PBS, resuspended in PBS containing 50 μg/ml PI and 50 μg/ml RNase A. The DNA content of each cell nucleus was determined by flow cytometry. 300 cells were counted mixed with MethoCult medium (StemCell, Vancouver, BC, Canada) and seeded in 3.5 cm dish. Clones were counted and diameter of each clone was measured under microscope after culture for 7 days.
7
Purification and Transduction of Primitive Hematopoietic Cells
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Cells were labeled with anti-human LIN cocktail (anti-CD3/CD14/ CD16/CD19/CD20/CD56), anti-CD15, anti-CD34, anti-CD38, anti-CD45RA (Biolegend) and anti-CD90 (BD Biosciences), antibodies specification described above, to perform purification of Primitive subpopulations (Lin- CD34+CD38- CD45RA- cells). The stained samples were FACS purified with the BD Aria cell sorter (BD Biosciences), achieving purity ranging between 92% and 99%.
Sorted primitive (HSC+MPP) populations were transduced as described above in “In vitro transduction of HD and WAS patients’ derived BM or MPB CD34+ cells” section. After transduction, cells were collected, washed, and transplanted in NSGW41 mice. An aliquot of cells was cultured in Iscove’s modified Dulbecco’s medium (IMDM), 10% fetal bovine serum (Cambrex, East Rutherford, NJ, USA) with stem cell factor (SCF), FLT3-L thrombopoietin (TPO) and IL-3 (all from Preprotech) at 20 ng/ml concentration (liquid culture) and harvested after 15 days to perform VCN estimation. An additional aliquot was used to perform Colony Forming Cell (CFC) assay according to the manufacturer’s procedure in Methocult medium (Stem Cell Technologies, Vancouver, Canada). At day 14, colonies were scored, singly picked and analyzed to evaluate the percentage of transduction.
Sorted primitive (HSC+MPP) populations were transduced as described above in “In vitro transduction of HD and WAS patients’ derived BM or MPB CD34+ cells” section. After transduction, cells were collected, washed, and transplanted in NSGW41 mice. An aliquot of cells was cultured in Iscove’s modified Dulbecco’s medium (IMDM), 10% fetal bovine serum (Cambrex, East Rutherford, NJ, USA) with stem cell factor (SCF), FLT3-L thrombopoietin (TPO) and IL-3 (all from Preprotech) at 20 ng/ml concentration (liquid culture) and harvested after 15 days to perform VCN estimation. An additional aliquot was used to perform Colony Forming Cell (CFC) assay according to the manufacturer’s procedure in Methocult medium (Stem Cell Technologies, Vancouver, Canada). At day 14, colonies were scored, singly picked and analyzed to evaluate the percentage of transduction.
8
Colony-Forming Unit Assay for Murine Hematopoiesis
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CFU assay was performed using buffers, medium, and dishes from StemCell Technologies (MethoCult GFM3434). Mouse blood was collected using a heparin-coated syringe and was placed in tube containing heparin (100 mg/mL, Merck). Spleens were subsequently collected and placed in Iscove’s modified Dulbecco’s medium (IMDM) medium supplemented with 2% FBS (StemCell Technologies). Spleens were minced with surgical scissors and smashed on a 40 µm cell-strainer in order to obtain single-cell suspension. Splenocytes were washed twice with IMDM medium containing 2% FBS, resuspended in IMDM medium, and counted using a Neubauer hemocytometer. Whole blood was treated with nine volumes of ammonium chloride solution (StemCell Technologies). Thereafter, blood cells were washed twice with IMDM medium and counted again. 2 to 3 × 105 splenocytes and erythrocyte-depleted blood cells were resuspended into Methocult medium and plated in Smart 6-well plates (StemCell Technologies). The number of colonies formed was assessed after 12 d of incubation at 37 °C, 5% CO2, and ≥ 95% humidity.
9
Quantifying Hematopoietic Colony Formation
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One and two months after IR, 1 × 105 BM cells were collected from each group and cultured for colony-forming units in MethoCult medium (StemCell Technologies, Vancouver, Canada). Then, the colonies were counted on day 7, as described previously.
10
Mouse Hematopoietic Colony Assay
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Femurs were isolated from 3–6 M-old mice (BM cells) or 11–12 M-old mice (spleen cells). Cells were plated in methyl cellulose MethoCult medium (M3434, M3234, STEMCELL Technology, Vancouver, Canada), as per the manufacturer’s instructions. For BFU-E, CFU-GM, and CFU-GEMM, 1.0×104 BM cells or 4.0×105 spleen cells obtained from a mouse were cultured in two 35 mm dishes in the presence of SCF, IL-3, IL-6, and EPO (M3434) for 12 or 13 days before observation. For CFU-E, 6.8×105 BM or 4.0×105 spleen cells obtained from a mouse were cultured in three 35 mm dishes containing MethoCult medium (M3234) supplemented with 10 U/mL rhEPO for 2 days before observation. BM cells were not treated to lyse red blood cells. Images of the entire 35-mm dish field were obtained using the image-stitching function of the BZ-X800 microscope (Keyence, Osaka, Japan). Colonies were counted by an observer who was blinded to the genotypes.
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