Pe conjugated rat anti mouse ifn γ antibody

A previously described procedure was followed for intracellular IFN-γ staining [27 (link)]. The splenocytes were cultured with different stimulants and controls (as described above in culture of splenocytes) for 10 h in a humidified incubator with 5% CO₂ at 37°C. Brefelin A (Golgistop; Pharmingen) was added to the wells and the plates were incubated for another 6 h. Cells from each treatment were suspended in FACS buffer (PBS containing 1% BSA and 0.2% sodium azide). They were incubated for 30 min on ice with appropriately diluted FITC-conjugated CD8 antibody (BD Pharmigen, clone 53–6.7) and APC-conjugated CD4 antibody (BD Pharmigen, clone L3T4-RM 4.5). Following three washes with FACS buffer, the cells were stained for intracellular IFN-γ with PE-conjugated rat anti-mouse IFN-γ antibody using the cytofix/cytoperm (Pharmingen) according to the manufacturer’s instructions. As an isotype control, cells stained with PE-conjugated rat IgG1 antibody were used. The cells were acquired on BD FACS Canto II Flow cytometer (BD Biosciences, CA, USA). The data were analyzed using BD FACSDIVA version 6 software (BD Biosciences, CA, USA) and the proportion of CD4⁺ and CD8⁺ T cells that secreted IFN-γ was determined.