Methocult h4434 classic medium

In the experiments to assess the role of PKCδ or ROCK1/2 on IFNα-mediated anti-clonogenic effects, primary PBMCs from PV patients were transfected with control or PKCδ (ON-TARGETplus Human PRKCD siRNA SMARTPool, # L-003524-00-0005, Dharmacon), or ROCK1 and ROCK2 siRNAs using TransIT-TKO Transfection Reagent (Mirus), as per the manufacturer’s instructions, as indicated. Hematopoietic progenitor colony formation for human erythroid precursors (burst-forming unit erythroid [BFU-E]) was then determined in clonogenic assays in MethoCult H4434 Classic medium (Stemcell Technologies) in the absence or presence of human IFNα (1000 IU/mL). In the experiments to assess the effects of drug-targeted inhibition of ROCK1/2 on IFNα-mediated anti-clonogenic effects, HEL cells were seeded in MethoCult H4534 Classic medium (Stemcell Technologies) and treated with vehicle-control (DMSO), GSK429286A (5 μM), and/or human IFNα (100 IU/mL). In parallel, primary PBMCs from PV patients were seeded in MethoCult H4434 Classic medium (Stemcell Technologies) and treated with vehicle-control (PBS), Fasudil (30 μM), and/or human IFNα (1000 IU/mL). Hematopoietic colony formations were scored as in previous studies^{21 (link),71 (link)}. Percent (%) of colony formation was calculated by dividing each colony count by the average colony count in the control group, and multiplying by 100%.

Colony formation assays in MethoCult™ were performed after a 4 hour cytolysis reaction with an E : T ratio of 10 : 1 PBMCs to target cells and 1 nM triplebody Her2-3-Her2 or 33-3-19. Colony Forming Cells (CFCs) were detected and counted using the MethoCult™ H4434 Classic medium (Stem Cell Technologies, Munich). Briefly, cells were harvested and washed with Iscove's Modified Dulbecco's Medium after the cytolysis reaction. 5,000 to 10,000 target cells were seeded to 1 mL of rigorously vortexed MethoCult™ medium and transferred to a 24-well tissue culture plate. The sample well was surrounded with water-containing wells and the dish was incubated at 37°C/5 % CO₂ for 7 days. On day seven, 100 μL of a 1 mg/mL iodonitrotetrazolium chloride (INT) solution in PBS was added and after an overnight incubation at 37°C/5 % CO₂ the number of colonies was counted.

Cells were seeded into MethoCult H4434 Classic medium (StemCell Technologies) with the addition of 2.5 μg/ml puromycin. Cultures were incubated at 37 °C in a humidified atmosphere of 5% CO₂. The colonies were replated every 7 days under the same conditions.