Cytokine and Chemokine Expression Analysis

We examined the Il-23, Il-1β, Ccl2, Ccl3, Ccl4, Inos, and Gapdh expression in differentiated- (1 × 10⁷ cells) and undifferentiated-MPRO clone 2.1 cells (2 × 10⁷ cells). Both differentiated- and undifferentiated-MPRO clone 2.1 cells were treated with the supernatants of Cl66, Cl66-Dox, Cl66-Pac, and SF media cells for 24 h. Details of RNA isolation and reverse transcription are described in [54 (link)]. We prepared qRT-PCR reactions using PowerUp™ SYBR™ Green Master Mix (Thermo Fisher, Carlsbad, CA, USA), cDNA, gene-specific primers, and nuclease-free water. The results were analyzed using Thermo Fisher Connect (Thermo Fisher, Carlsbad, CA, USA). Mean Ct values of the target genes were normalized to mean Ct values of the endogenous control, Gapdh; [−∆Ct = Ct (GAPDH) − Ct (target gene)]. We calculated the ratio of mRNA expression of target genes versus Gapdh (2^(−∆Ct) and further normalized it with the control (MPRO cells in SF) (2^{(−∆∆Ct)}). Melting curve analysis was performed to check the specificity of the amplified products. The details of the sequence of gene-specific primers are in Table 1.