Supplies: Ethanol 100 USP (highest quality) FACS Staining Buffer (1XPBS w/ 3 calf serum, 0.05 azide--filtered) ―Dilute staining antibodies in Buffer DNAse (Sigma D-5025, Bovine Pancreas) RNase (Boehringer, 25 mg bovine pancrease) Anti-BrdU-FITC (Becton Dickinson or Phoenix Flow) 0.15 M NaCl, 1.5 M NaCl 10 Paraformaldehype (kept as stock in -80°C) Tween 1M MgCl2 FACS Tubes

Protocol:

Cells in 96 well FACS plate

Block with 24G-2 Surface stain cells as usual -Omit fourth channel labeled antibodies on all stains; EtOH destroys APC Prepare tubes from which to transfer EtOH drop wise (1.2 ml EtOH on ICE) Resuspend cells from 96 well place with 100 µl 0.15M NaCl (cold) Transfer to FACS tubes ON ICE. Add 400 µl 0.15M NaCl to each tube Vortex at 1/3 speed and add EtOH with pasteur pipette at 1 drop per second. This is a critical step... do not add EtOH too quickly Incubate on ice for 30 minutes Spin 10 minutes @ 2000 RPM, 4° C Dump and shake liquid into waste Using repeat pipetter, squirt 1 ml FACS staining buffer into each tube Spin 10 minutes and dump as before (step 8) Add 1 ml 1 paraformaldehyde + 0.05 Tween 10 -For 20 ml: 2.0 ml 10 paraformaldehyde 10 µl Tween-20 Incubate at room temperature for 30 minutes Incubate on ice for 30 minutes Spin and dump as before (step 8) Add 1 ml DNAse (0.15M NaCl + 4.2mM MgCl + 100 Kunitz units/ml DNAse) -For 50 ml: 46.5 mL dH20 200 ul MgCl2 (1M stock) 1500 uL NaCl (5M stock) 100 Kunitz units Dnase (volume depends on activity of batch) Incubate for 30 minutes @ 25° Spin 10 min. and dump as before (step 8) Transfer cells from FACS tubes to 96-well plate. Wash once with staining media. Block with 10 rat serum. Incubate 15 minute on ice. Spin and dump as before (step 8: it is critical to spin at high speed once the cells have been fixed with EtoH/ PFA since they become less dense). Add anti-BrdU-FITC or biotin (1:20 dilution for Phoenix flow). Pipette up and down to resuspend pellet. Incubate for 30 minutes on ice (or overnight at 4°C). Wash and dump as before. Transfer cells into FACS tubes.