Krc0121

Cytokine levels were estimated in the serum samples using rat ELISA kits from Thermo Fischer Scientific/Invitrogen/Life Technologies, USA. The kits used were IL-1β (Invitrogen BMS630), TNFα (Life Technologies IVGN ERA56RB), IL-6 (Life Technologies IVGN ERA31RB), and IL-12p70 (Invitrogen KRC0121). Briefly, 100 uL of 1× coating buffer and antibodies of interest were added to Nunc MaxiSorp 96 well plates and incubated for 16 h at 4 °C. The following day, contents were discarded and washed three times with 1× PBS in 0.1% tween 20 (PBS-T). 200 uL of 1× blocking buffer was added to the wells and the plates were incubated at RT for an hour. Recombinant standards and samples were then added in duplicates in serial dilutions (only for standards) as described in the manufacturer’s protocol. Standards and samples were incubated for 2 h at RT. After 3 washes with PBS-T, detection antibody was added and incubated for 1 h at RT followed by 3 washes. HRP-avidin was added and left in the dark for 30 min at RT. After 5 washes, 100 uL of 1× TMB was added to all the wells. After 15 min, 50 uL of 2 N H₂SO₄ was added to each well. Plates were immediately read at 450 nm using SpectraMax M2 Gemini Molecular Device Microplate Reader and the values were processed for statistical analysis.