4.5. qPCR Analysis to Determine Relative Cytokine mRNA Expression in the Colon Tissue and in Peritoneal Macrophages

To obtain enough cells from the colon tissue samples and to perform all the analysis, we pooled the colon tissue samples from five (5) male mice into one and the colon tissue samples from five (5) female mice into one for each group. Thus, we had only two samples (n = 2) of colon cells from each group of animals. The colon tissue was treated with 1 mL Trizol reagent (Sigma Aldrich, St. Louis, MO, USA) and total RNA was isolated using Direct-zol RNA miniprep plus kit (Zymo research Corp, Irvine, CA, USA). One microgram of this isolated RNA was reverse transcribed to cDNA using a Readyscript cDNA synthesis kit (Sigma Aldrich, St. Louis, MO, USA).

Before collecting the colon tissue from the euthanized mice, we washed the peritoneal cavity of each mouse with 1 mL sterile saline solution and the peritoneal fluid was carefully collected into a sterile tube. Peritoneal macrophages were collected from each mouse by incubating the peritoneal fluid in 6 well culture plates at 37 °C and 5% CO₂ for 4 h. After removing the non-adherent population (processed separately for B1 cells flow cytometry), the plate was washed three times with sterile RPMI medium. The cells were then lifted from the plates, counted and plated again on a 24-well culture plate at a concentration of 1 × 10⁶ cells/well. The cells were then incubated for 24 h in RPMI 1640 media supplemented with 10% fetal bovine serum. Both culture supernatant and cells were collected separately. Trizol reagent was added to the cells and total RNA was isolated from the adherent cells using Direct-zol RNA miniprep plus kit (Zymo research Corp, Irvine, CA, USA). Isolated RNA (400 µg) was used as a template to perform reverse transcriptase PCR reaction using Readyscript cDNA synthesis kit.

IL-6, IL-10, RORγ, TNF-α, GATA-3, TBX21, and IL-17a genes were qPCR amplified, from the colon tissue cDNA prepared above. Similarly, TNF-α, IL-6, and IL-10 genes were qPCR amplified from the peritoneal macrophages, using the pre-optimized TaqMan Gene expression Assays and TaqMan^® multiplex master mix (Thermo Fisher Scientific, Rockford, IL, USA). GAPDH was included in the assay as a housekeeping gene. PCR amplification was carried out on QuantStudio 6 Flex Real-Time PCR system and the amplification data was analyzed using the QuantStudio Real-Time PCR software version 1.1 (Thermo Fisher Scientific, Rockford, IL, USA).