4.3 (b) Expression profile of TLRs and HLA-DR in response to Rv2615c
The expression profile of TLR2 and TLR4 was evaluated through flow cytometry following 24h stimulation of THP-1 macrophages with the 10µg/ml Rv2615c protein. The controls included were TLR4 agonist- LPS (40ng/ml) and TLR2 agonist- Cell Wall Fraction of H37Rv (CWF) (5μg/ml) (BEI Resources) (Manček-Keber and Jerala, 2015). Following incubation, cells were harvested and washed in PBS and stained with APC labelled anti-human TLR2 (CD282) antibody (Thermo Scientific) or APC labelled anti-human TLR4 (CD284) antibody (Thermo Scientific) and PE labelled anti-human HLA-DR antibody (Thermo Scientific). Cells were analysed in BD Accuri C6 flow Cytometer. Results were analysed on the basis of percentage of positively stained cells.
For inhibitor assay, 1h prior to stimulation; cells were blocked with Anti-TLR4 monoclonal antibody (Thermo Scientific) and analysed for expression of TLR4 and HLA-DR after 24h using flow cytometry.