dust mate

Can not you perform intracellular staining after culture of whole popliteal LN cells instead of purification of DCs? I am not sure even with CD11c MACS you will achieve high purity anyway. these beads work well when initial cell frequency is high (e.g. in the spleen after 2 rounds of AutoMACS I was getting around 60-70% CD11c+ cells (which is low) when initial frequency was 1-3% only. but when initial frequency was around 30% then purity was >97%).

prepare single cell suspension first and then use no more than 2-3ml buffer per spleen. Don't lyse more than 1 minute! This time is more than enough