Measuring CCL5 Expression and Cytokine Levels

Total RNA was extracted using RNeasy mini kits (Qiagen, Valencia, CA) as described in the manufacturer's protocol. Real time reverse transcriptase polymerase chain reactions (RT-PCR) using Bio-Rad iCycler was performed to determine the mRNA expression levels. The reaction conditions included reverse transcription at 50°C for 30 min, denaturation at 95°C for 15 min, followed by amplification for 45 cycles (95°C for 15 sec, 54°C for 30 sec, and 76°C for 15 sec). The CCL5 primers are 5′ ACC AGT GGC AAG TGC TCC A-3′ for forward and 5′ ACC CAT TTC TTC TCT GGG TTG GCA-3′ for reverse (Integrated DNA Technology, Coralville, IA). Separate hypoxanthine-guanine phosphoribosyl-transferase (HPRT - house-keeping gene) amplification was used to normalize gene expression. The data was analyzed using the equation 2^−ΔΔCT method. The primer sequences and PCR conditions for p38 and Akt isoforms used in this study and the annealing temperatures were based on our previous study30 (link).
Cell culture supernatants were collected and the cytokine protein concentrations were analyzed by a multi-cytokine bead assay system (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. The protein expression was measured using Bio-Plex Manager 5.0 software and 5PL standard curve.