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Percoll for PMN-Isolation

Percoll for PMN-Isolation - Protocols and Methods Forum

Percoll for PMN-Isolation - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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Old 05-23-2013, 01:03 PM
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Default Re: Percoll for PMN-Isolation

Purification of polymorphonuclear cells (PMNs) : dilute 200 - 400 Ál whole blood in EDTA-K3 up to 4 ml with NaCl 0.9% and centrifuge at 450 g for 10 mins. Resuspend pellet in 1 ml HBSS containing 0.2% EDTA. Stratify this suspension up 3 Percoll layers at different concentration (75%, 67% and 52%) such as described in specific article (F. Zagami : Phagocytic index - [Only registered users see links. ]) diluting Percoll in HBSS (100% Percoll = 9 parts Percoll and 1 part HBSS 10x) and then centrifuged at 1500 g for 30 mins. The neutrophils have been harvest between 67 and 75% gradients, after having attentively removed the cells from the superior phases. The harvest PMNs has been washed for 2 times with HBSS, and then submitted to purification of the possible erythrocytes contamination through a hypotonic lysis procedure. To the purpose the pellet has been resuspended in 0.5 ml dH2O and incubate for 35 sec. at RT and osmolarity is restored by adding 0.5 ml 1.8% NaCl solution. Suspension was immediately gently mixed and centrifuged at 1000 g for 10 min., and further washing in HBSS and therefore resuspended in Nutritive Salt Buffer (NSB - dissolve 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 10 mM HEPES at pH 7.2 and 8.9 mM glucose) and used within 6 hrs by the collecting.
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