I'm trying to use ACK lysis buffer to lyse RBCs from whole blood samples after an initial lysing with H2O (restoring isotonicity with 10% NaCl). My last step is using RIPA buffer + protease inhibitors to lyse the WBCs and use the protein lysate for western blotting. 

I am concerned with keeping the nuetrophils from activating before I lyse them at the end with RIPA, so I'd like to keep all my spins and reagents cold. I can't find any information about whether this might be an issue for the ACK lysis buffer step - would using cold lysis buffer make it any less effective? 

Thanks for the help!

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