ACK Lysis Protocol

Blood should be collected in heparinized micro-hematocrit capillary tubes (Fisher cat #22-362-566). The blood samples should be transferred to microtainer tubes containing EDTA (Becton-Dickinson cat #35973).

You can process the samples yourself by following the protocol below, or you can bring the samples directly to us in the microtainer tubes. Regardless, please phone us at 362-9348 if you desire this service as we need to coordinate both control bleeds (WT C57BL/6J and B6-GFP mice) and FACS time.

ACK Lysis Buffer (1L)

Component	Amount	Final Concentration
NH4Cl	8.3 g	0.15M
KHCO3	1.0 g	10mM
EDTA	200 ul 0.5M	0.1mM

add ddH2O to 800 ml, adjust pH to 7.2 - 7.4, finalize volume to 1L with ddH20

FACs Buffer (in 1L PBS)

Component	Amount	Final Concentration
FBS	20 ml	2% (v/v)
EDTA	2.0 ml 0.5M	1mM

Processing Blood Samples

• Take 100 - 150 µl blood from microtainer and transfer to an Eppendorf tube.

• Add 1.0 ml ACK Lysis Buffer to lyse the red blood cells.

• Vortex briefly and spin 5 minutes at 0.5 RCF at room temperature.

• Aspirate the buffer from the white cell pellet carefully with a p200 tip attached to the aspirator. There will be a ring of red blood cells surrounding the white cellular pellet. Try to remove this as well. If necessary, add another 1.0 ml of ACK Buffer, mix, spin, and remove additional cellular excess.

• Harvest cell pellet in 0.5 ml FACs buffer, transfer to 3 ml FACs compatible tube, and perform analysis.