Determination of the effect of CpG ODN1826 on IFN-Ɣ stimulated nitric oxide production in Raw 264.7 cell

Abstract

Aim: Determination of the effect of ODN1826 CpG on IFN-Ɣ-stimulated NO production in Raw 264.7 cells Materials and methods: This study was carried out with the support of the Cell Culture Laboratory of the Institute of Bio-Medical Sciences of MNUMS, and the Clinical Pathology Laboratory of the Department of Immunology of Bio-Medical School. Raw 264.7 cells were cultured in a 96 well plate with a volume of 100 μl and 10,000 cells per well for 24 hours at 37ºС; 0.5% CO2 incubator. Experimental groups include 1. PBS (negative control) 2. IFN-Ɣ (positive control) 3. IFN-Ɣ + ODN1826. Incubation with treatment substances at 37ºС; 0.5% CO2 incubator for 24, 48, and 72 hours, 50 μl of the cell culture supernatant was taken and NO yield was the determined by Greiss method. The cells were lysed using RIPA lysis buffer, and the protein concentration was determined using the BCA method. SDS gel electrophoresis was performed to separate protein molecular weights, and the gel was stained with Coomassie Blue dye. Results: In the experiment, the nitric oxide (NO) output in RAW cells at 24, 48, and 72 hours increased consecutively in all groups. When comparing the results at 72 hours, all groups exhibited statistically significant increases in Nitric Oxide output compared to the negative control. In comparison with the positive control and the IFN-Ɣ induced group, ODN1826 significantly enhanced nitric oxide production (p < 0.001). The protein was run on an SDS gel and stained with Coomassie Blue, revealing the iNOS protein. Conclusion: ODN1826 CpG increases nitric oxide production and induces iNOS protein synthesis.