Mononuclear cells (MNCs) from the lungs were prepared as described previously (Alghetaa et al., 2018 (link); Mohammed et al., 2020b (link); Alghetaa et al., 2021 (link); Sultan et al., 2021 (link)). Briefly, excised lungs were subjected to a mechanical tissue homogenizer (Seward, England). The tissue suspension was filtered and the single-cell mixture was suspended in cold fluorescence-activated cell sorting buffer (FACS) prepared from PBS enriched with 10% fetal bovine serum protein (FBS). RBC-lysis buffer (Sigma-Aldrich, United States) was used to remove the RBC from the mixture before being filtered with a 100 micron-strainer (Fisher Scientific, China). The filtered suspension was layered on Ficoll gradient, Histopaque-1077 (Sigma-Aldrich, United States) to separate MNCs. All MNC preparations were finally suspended in FACS and cell counts were measured by using trypan blue dye via auto cell counter 2000 (Bio-Rad, United States). Spleen cell preparations were carried out in a similar fashion except that they were not subjected to Ficoll gradient separation.
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