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Leukocyte Isolation

TH Taylor A. Heim
AS Austin C. Schultz
ID Ines Delclaux
VC Vanessa Cristaldi
MC Madeline J. Churchill
AL Amanda W. Lund
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Dorsal and ventral sides of ears were separated and incubated for 30 minutes at 37 C in 1ml of HBSS (Hyclone) containing CaCl2 and MgCl2 supplemented with 125 U/ml of collagenase D (Invitrogen) and 60 U/ml of Dnase-I (Sigma-Aldrich). LNs, spleens or digested skin were smashed on a scored plate and put through a 70µm cell strainer to generate single cell suspensions. Spleens were resuspended for 2 minutes in 2ml of ammonium-chloride-potassium lysis buffer.

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