Guava easycyto flow cytometer

Cells were collected from BALF and resuspended in a buffer containing 154 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA for red blood cell lysis for 5 minutes. After washing with PBS containing 2% FBS, cells were stained with anti-CD45.2 (Biolegend; 109808), anti–Siglec F (Invitrogen; 12-1702-82), anti–Gr-1 (BioLegend; 108412), anti-CD4 (Biolegend; 100516), anti-CD8a (Biolegend; 100708), anti-Mac-1 (CD11b) (Biolegend; 101208), anti-CD11c (Biolegend; 117307), anti-NK-1.1 (Ly-55) (Biolegend; 108707), anti-B220 (Biolegend; 103211), or anti-IgG2a (Biolegend; 407109) antibodies for 30 minutes on ice. To confirm the Th2 cell differentiation, cells were collected from BALF and fixed with 1% paraformaldehyde (PFA) and permeabilized with PBS containing 0.1% Triton X-100 and 0.2% bovine serum albumin for 3 minutes. The cells were then incubated with PE-conjugated anti-GATA3 (Invitrogen; 12-9966-42) and APC-conjugated anti-CD4 (Biolegend; 100516) antibodies for 1 hour. Flow cytometry analysis was performed using a Guava easyCyto flow cytometer (Merck Millipore).

Cells were collected from bronchoalveolar lavage fluid (BALF) and resuspended in RBC lysis buffer (154 mM NH₄Cl, 10 mM KHCO₃, and 0.1 mM EDTA) for 5 min. After washing the cells with PBS containing 2% fetal bovine serum (FBS), the cells were stained with either anti-CD45.2 or anti-IgG2a antibodies on ice for 30 min. Flow cytometry was performed using Guava easyCyto flow cytometer (Merck Millipore).