Ctl ultimate s6 immunospot analyzer

96-well plates (Immunlon 2HB) were coated with 50 μg/mL PLP in 100 μL of PBS and incubated overnight at 4°C. The PLP coating solution was discarded and the plate was blocked with PBS containing 0.5% FBS for 1 hour prior to addition of splenocytes. Immediately after their isolation, splenocytes were seeded onto the coated 96-well plate at a cell density of 1×10⁶ cells per well in a final volume of 100 μL. The splenocyte cultures were incubated for 48 hours at 37°C in a CO₂ (5%) incubator. Following incubation, each plate was washed 4x with PBS containing 0.05% Tween 20 for 2 minutes, followed by washing 2x with PBS for 2 minutes. 100 μL of horseradish peroxidase (HRP) conjugated anti-mouse IgG at a concentration of 1 μg/mL was added to each well and incubated for 1 hour at 37°C. Following a second wash step, identical to previously described, a buffer containing TrueBlue Peroxidase Substrate (Kirkegaard & Perry Laboratories, Inc) and agarose (1:1 ratio) was heated in a water bath to 56°C and 100 μL was added to each well using reverse pipetting to avoid bubbles. Plates were incubated overnight at 4°C before reading on the CTL Ultimate S6 ImmunoSpot Analyzer (Cellular Technology Limited). Analysis of spots was done using CTL ImmunoSpot software (Cellular Technology Limited).

Immulon 2HB 96-well ELISPOT plates (Thermo Fisher Scientific, Waltham, MA) were coated with PLP antigen at a concentration of 50 μg/ml in 100 μL PBS, incubated overnight at 4°C, then blocked with cRPMI for 1 hour at 37°C. Splenocytes isolated from healthy controls and EAE mice on day 25 following in vivo treatment were seeded at 1×10⁷ cells/mL (1×10⁶ cells/well) in the coated and blocked plates and incubated (37°C, 5% CO₂) for 48 hours. Plates were then washed 4X with PBS-Tween (containing 0.05% Tween 20) and 2X with PBS for 2 minutes each. Horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody in PBS containing 0.5% FBS was added to each well (100 μL) at a concentration of 1 μg/mL and incubated at 37°C for 1 hour. After a second wash with PBS-Tween and PBS, detection buffer containing a 1:1 ratio of TrueBlue Peroxidase Substrate (Kirkegaard & Perry Laboratories, Inc) and agarose heated to 56°C was added to each well (100 μL) and incubated at 4°C overnight. Plates were read on a CTL Ultimate S6 ImmunoSpot Analyzer (Cellular Technology Limited) and analyzed using CTL ImmunoSpot software (Cellular Technology Limited).