Neutrophil Purification from Murine Peripheral Blood

 

  1. Obtain whole blood drawn into preservative-free heparin containing plastic syringes and keep at room temp

 

  1. Dilute blood with at least 1 volume of PBS with 0.5% albumin (PBS/HSA)
    1. I use clinical grade human serum albumin

 

  1. Immediately prior to spin prepare a discontinuous Histopaque gradient with all components equilibrated to room temp
    1. 5ml 1.119
    2. 1.5ml 1.083
    3. 1.5ml 1.077

 

  1. overlay diluted blood

 

  1. spin 30min 1800rpm (700xg) room temp NO BRAKE

 

  1. the mononuclear cells are at the 1.077/1.083 interface

 

  1. the granulocytes are at the 1.083/1.119 interface

 

  1. remove granulocytes to PBS/HSA and rinse
    1. I do in 50ml conical
    2. Spin 10min 1500rpm

 

  1. Lyse rbc’s in 36ml sterile irrigation water for 30” and rapidly buffer with 9ml 5X PBS/HSA and spin to pellet cells

 

  1. resuspend in PBS/HSA to no more than 5 x 106 cells/ml