. Author manuscript; available in PMC: 2012 Mar 1.

Published in final edited form as: Nat Protoc. 2011 Feb 17;6(3):296–313. doi: 10.1038/nprot.2010.184

Table 4.

Troubleshooting

Step	Trouble	Reason	Trouble shootings
Step 15	Spontaneous hPSC differentiation, loss of pluripotency	Feeder-layer cells of low quality or inadequate density	- MEF lots have to be pretested for their ability to support hPSC growth. Make sure to use a semiconfluent MEF monolayer
Step 15	Spontaneous hPSC differentiation, loss of pluripotency	Feeder-layer cells of low quality or inadequate density	- Mark differentiated colonies with objective markers under a microscope and eliminate differentiated hPSC colonies by aspiration of the marked area with glass Pasteur pipettes during medium change.
Step 23	High spontaneous adipogenesis in OP9 cultures or loss of homogeneity of OP9 with formation of cord-like structures (Fig.4)	- Adipogenic serum	- Select FBS lots with minimal adipogenic effect.
		- Incorrect split ratio	- Adjust the split ratio with a new FBS lot to achieve confluence by day 4 of differentiation.
		- Incomplete enzymatic dissociation of OP9 cells during passage	- Make sure OP9 cells are completely detached after trypsinization.
Step 46	Low efficiency of hematopoietic differentiation	- hPCSs spontaneously differentiate in maintenance culture	- See troubleshooting for step 15
		- Massive adipogenesis in OP9 cells	- See troubleshooting for step 23
		- Non-optimal plating density of hPSCs	- Optimize plating density using an initial range of 0.5×10⁶ cells per 10 cm dish with a 0.5×10⁶ interval.
		- Low density of OP9 monolayer used to initiate differentiation of hPSCs	- Make sure to use overgrown OP9 cells before plate hPSCs. OP9 cells should be cultured for additional 4–6 days after forming a confluent monolayer.
		- Irregular feeding	- Strictly adhere to the feeding schedule described in this protocol.
Step 79	Number of isolated CD45⁺ cells is low	- Low efficiency of hematopoietic differentiation	- See troubleshooting for step 46.
		- Loss of cells during magnetic separation	- Avoid bubble formation in column during separation procedure by using degassed MACS separation buffer.
			- Remove cell aggregates by filtration before applying cell suspension to the column.
			- Make sure that the primary antibodies have been appropriately titrated.