Nalm6, Jurkat, and 658w WT and casp-3/7−/− cells were plated in 3 ml of RPMI 1640 complete medium in six-well plates at a concentration of 2 × 105 cells per well. The cells were then treated for 24 to 48 hours with either Birinapant (50 nM) and TNF (10 ng/ml), ABT263 (5 mM), or vehicle. The cells were then collected, washed with PBS, mixed with Methocult Optimum media (catalog no. 4034; StemCell Technologies), and plated at a concentration of 1000 cells per well in six-well SmartDish plates (catalog no. 27370; StemCell Technologies). Cells were aliquoted in duplicate samples. After 10 to 14 days, colony-forming units were visually counted under a microscope.
Methocult optimum media
Manufactured by STEMCELL
Methocult Optimum media is a methylcellulose-based medium designed for the enumeration and culture of human and mouse hematopoietic progenitor cells from bone marrow, cord blood, and mobilized peripheral blood samples.
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Lab products found in correlation
3 protocols using methocult optimum media
1
Clonogenic Assay for Apoptosis
2
Hematopoietic Potential of CMS iPS-Derived CD34+ Cells
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FACS-sorted iPS-derived CD34 + cells from the CMS and non-CMS subjects were plated at a density of 105 cells per 35 mm dish combined with MethoCult Optimum media (Stemcell Technology, Cat. H4034) and 2% FBS (Sigma, Cat. F2442). Dishes were incubated at 37 °C in an incubator with 5% CO2 and 5% O2 for 14 d, at which time colonies were scored for BFU-E and CFU–GEMM (granulocyte, erythrocyte, monocyte, megakaryocyte). For EPO dose-response experiments, Methocult classic (without EPO) (Stemcell Technology, Cat. H4434) was mixed with various doses of EPO (0.5, 1, 1.5, 2, 2.5, 3 and 3.5 units) and assayed for colony production. We used N = 4 subjects for each group that was tested by this assay.
3
Colony Formation Assay Protocol
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At 48 h post gene editing, 250 cells per well were plated in Methocult Optimum media in SmartDish plates (both StemCell Technologies). Plates were incubated in a secondary enclosure at 37 °C, 5% CO2, and 5% O2 for 14 d before scoring colonies using the human mPB program on a STEMvision imager (StemCell Technologies).
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