Isolation of lymphocytes from lung tissues

TK Toshio Kanno
TN Takahiro Nakajima
SY Satoru Yokoyama
HA Hikari K. Asou
SS Shigemi Sasamoto
YK Yasuhiro Kamii
KH Koji Hayashizaki
YO Yasuo Ouchi
TO Taishi Onodera
YT Yoshimasa Takahashi
KI Kazutaka Ikeda
YH Yoshinori Hasegawa
YK Yuki Kinjo
OO Osamu Ohara
TN Toshinori Nakayama
YE Yusuke Endo
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Mice were euthanized by anesthesia and their lungs were perfused with 5 mL cold PBS solution through the right ventricle. Lungs were transferred to a tube containing ice-cold digestion buffer [RPMI 1640 supplemented with collagenase type III (200 U/mL; Worthington) and DNase I (200 μg/mL; Sigma-Aldrich)]. Individual lungs were dissociated in 3 mL digestion buffer using a GentleMACS tissue dissociator (Miltenyi Biotec). This was followed by incubation for 30 min at 37 °C with frequent agitation and a final GentleMACS dissociation. Lung mononuclear cells were separated by centrifugation on Percoll (GE Healthcare).

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