STEMdiff™ Hematopoietic Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
- Throughout the protocol, warm all media to room temperature (15 - 25°C) before use. Do not leave media at room temperature for extended periods of time.	-Thaw STEMdiff™ Hematopoietic Basal Medium at room temperature (15 - 25°C) or overnight at 2 - 8°C. Mix thoroughly. -Thaw Supplement A or B at room temperature (15 - 25°C) or at 2 - 8°C until just thawed. Mix thoroughly. If necessary, centrifuge for 30 seconds to remove liquid from cap. -If it is difficult to anticipate the correct number of cells to plate (e.g. when testing a new hPSC line), use two or more numbers that differ by 2- to 3-fold (e.g. 5000 cells per dish and 10,000 cells per dish).

Upstream tips
- Throughout the protocol, warm all media to room temperature (15 - 25°C) before use. Do not leave media at room temperature for extended periods of time.
Protocol tips
-Thaw STEMdiff™ Hematopoietic Basal Medium at room temperature (15 - 25°C) or overnight at 2 - 8°C. Mix thoroughly. -Thaw Supplement A or B at room temperature (15 - 25°C) or at 2 - 8°C until just thawed. Mix thoroughly. If necessary, centrifuge for 30 seconds to remove liquid from cap. -If it is difficult to anticipate the correct number of cells to plate (e.g. when testing a new hPSC line), use two or more numbers that differ by 2- to 3-fold (e.g. 5000 cells per dish and 10,000 cells per dish).

Publication protocol

Improved and simplified differentiation of iPSCs to CD43+ primitive hematopoietic progenitor cells (HPCs) is achieved using Stem Cell Technologies STEMdiff™ Hematopoietic Kit (Catalog # 05310). On day − 1, feeder-free iPSCs that have been expanded in TeSR-E8 media are passaged with ReLeaSR (STEMCELL technologies) into mTeSR E8 medium with 0.5 μM Thiazovivin onto matrigel coated (1 mg/mL) 6-well plates (Corning Costar). Small aggregates of ~ 100 cells each are plated at 10–20 aggregates per cm2. The initial plating density is critical as higher density impairs mesoderm differentiation and lower density decreases yield. Thus one can plate iPSCs at 2–3 different densities and select the wells on day 0 that have optimal density to proceed with. When approximately two 100 cell colonies per cm2 have been achieved, replace TeSR-E8 medium with medium A (Basal medium plus Supplement A at 1:200 dilution, 2 mL per well of a 6-well). On day 2 (48 h after original media change), do not fully change media, but rather replace 50% medium A, 1 mL per well of a 6-well. On day 3, carefully remove all media by tilting the plate to one side and aspirating from the edge. Then add 2 mL/well medium B (Basal medium plus supplement B at 1:200). Without removing media, supplement with 1 mL/well of medium B on days 5, 7, 9. On day 10 and again on day and 12, non-adherent cells may be collected. To maintain purity, do not wash cells off the well, but merely remove medium with non-adherent cells carefully and centrifuge 300 x G 5 min. After centrifugation, replace conditioned medium back to each well and add 1 mL fresh medium B if further collection on day 12 will be completed.

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Manufacturer protocol

Download the product protocol from STEMCELL technologies for STEMdiff™ Hematopoietic Kit below.

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