Recombinant cytokines

All the recombinant cytokines, Abs, and ELISA reagents were purchased from BD Biosciences (San Diego, CA, USA) or unless mentioned. TLR-3L [poly (I:C); Catalog: tlrl-picw] was procured from Invivogen (San Diego, CA, USA).

Three mice from all the eight groups of batch-II were randomly selected, sacrificed and their spleens were removed aseptically. The cytokine estimation was performed using the method published earlier [43] (link). Briefly, single cell suspension of splenocytes was prepared by maceration of spleens. The splenocytes from each mouse (1×10⁶ cells/well) were suspended in a 24-well tissue culture plate in triplicates. The cultures were stimulated with particular antigen/s alone or in combination (5 µg/ml each antigen) corresponding to their designated groups or Concanavalin A (Con A, 5 µg/ml; Sigma, USA). The culture supernatants from the wells were collected after 48 h. The expression of cytokines i.e., TNF-α, IFN-γ, IL-2, IL-4 and IL-10 were measured by ELISA using BD OptEIA Kit, (BD Biosciences, USA) according to the manufacturer's instructions. The levels of cytokines were determined with the help of standard curves generated using recombinant cytokines (BD Biosciences, USA) and presented as picograms per millilitre (pg/ml).

Primary murine keratinocytes were transfected with siRNA-410 and siRNA-NC (20 μM) by TurboFect or NHEK were treated with different recombinant cytokines (all from BD) as indicated. Cells were harvested after 24 or 48 h and cold 70% ethanol was gently added drop-wise for fixation, followed by resuspension in a blocking solution (2% BSA, 5% FBS, 0.2% Triton X-100 and 0.1% sodium azide) and incubated at 4 °C for 10 min. Cells were then pelleted and resuspended in propidium iodide solution (0.1 mg ml⁻¹, Calbiochem/EMD). Flow cytometry was used to analyse cell cycle.