The aliquots of 106 splenocytes, isolated from each mouse, were suspended in a
buffer [0.1% NaN3, 1% FCS in PBS (pH 7.4)], exposed to appropriate antibodies, and
incubated at 4°C for 30 min in the dark. Thereafter, cells were washed, 10,000 events
acquired by FACSCalibur flow cytometer, and analyzed using CellQuest Pro software (BD
Biosciences, San Diego, CA), as previously described.4 ,16 For quantification of immune cell
markers, such as CD4 (TH), CD8 (TC), CD3 (T cells), CD4/CD25
(Treg), CD45RB220 (B cells), CD335 (NK cells) and CD11b (macrophages), the
following fluorochrome-labeled antibody clones were used: peridinin chlorophyll-a protein
(PerCP)-conjugated anti-CD4 (clone RM4–5), fluorescein isothiocyanate (FITC)-conjugated
anti-CD8 (clone 53–6.7), allophycocyanin (APC)-conjugated anti-CD25 (clone 3C7),
phycoerythrin (PE)-conjugated anti-CD3 (clone 145–2C11), APC-conjugated anti-CD45RB220
(clone RA3–6B2), FITC-conjugated anti-CD335 (clone NKp36), and PerCP-conjugated anti-CD11b
(clone M1/70) (all from BD Biosciences).
buffer [0.1% NaN3, 1% FCS in PBS (pH 7.4)], exposed to appropriate antibodies, and
incubated at 4°C for 30 min in the dark. Thereafter, cells were washed, 10,000 events
acquired by FACSCalibur flow cytometer, and analyzed using CellQuest Pro software (BD
Biosciences, San Diego, CA), as previously described.4 ,16 For quantification of immune cell
markers, such as CD4 (TH), CD8 (TC), CD3 (T cells), CD4/CD25
(Treg), CD45RB220 (B cells), CD335 (NK cells) and CD11b (macrophages), the
following fluorochrome-labeled antibody clones were used: peridinin chlorophyll-a protein
(PerCP)-conjugated anti-CD4 (clone RM4–5), fluorescein isothiocyanate (FITC)-conjugated
anti-CD8 (clone 53–6.7), allophycocyanin (APC)-conjugated anti-CD25 (clone 3C7),
phycoerythrin (PE)-conjugated anti-CD3 (clone 145–2C11), APC-conjugated anti-CD45RB220
(clone RA3–6B2), FITC-conjugated anti-CD335 (clone NKp36), and PerCP-conjugated anti-CD11b
(clone M1/70) (all from BD Biosciences).