Isolation and Identification of viable immune cells from murine lungs
Instrument: Precellys Evolution
Sample: Mouse whole lung (~0.3 g; fresh)
Protocol:
Collect the whole lung directly in the 7 mL CK28 (Cat no KT03961-1-302.7) Precellys® tube (or a bijou for the sieve hand grinding method) containing 1 ml sterile endotoxin-free PBS
Beat the organ in the Precellys® for 1 cycle of 10 s at 4500 rpm (RT)
Filter the homogenate with a 70 µm strainer to remove big debris and beads, rinse with an additional 1 mL PBS [OR 2’-3’) Push the lung through a 70 µm sieve using the plunger of a 5 ml syringe and rinse with 1 mL of PBS]
Centrifuge at 600 g for 10 min
Lyse the red blood cells (ACK buffer or alternative method)
After centrifugation, resuspend the cells in 0.5 ml PBS
Count the total and viable cells using trypan blue exclusion
Stain the cells for flow cytometry (here we used antibodies against CD45, CD11b, GR1, F4/80 and CD3).
Results:
While the yield of macrophages recovery is comparable to a standard manual method, the Precellys® Evolution leads to a higher yield of some immune cells (CD45+), notably neutrophils and lymphocytes.
from the Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, UK
