Hamster anti mouse cd3e

ELISpot was performed as previously described [35 (link)] and according to the manufacturer’s instructions (R&D Systems, SEL485 and SEL404, Minneapolis, MN, USA). Briefly, rPfCelTOS (10 µg/mL) was used as the stimulating antigen. Hamster anti-mouse CD3e (1 µg/mL [BD Biosciences, 553057, San Jose, CA, USA]) was used as a positive control for cell stimulation. The negative control was culture media in place of a stimulating antigen. Splenocytes were plated at 2 × 10⁵ cell/well. Plates were incubated for 48 h at 37 °C with 5% carbon dioxide. Spot counting was performed using an AID ELISpot Reader (Autoimmun Diagnostika, Strassberg, Germany).

CD4⁺ T-cells (1 x 10⁵ cells/well) were isolated using a CD4⁺ T-cell Isolation kit (130-104-454) (Miltenyi, Bergisch Gladbach, Germany) and cultured in a round-bottomed 96-well plate pre-coated with purified hamster anti-mouse CD3e (BD Biosciences, 553057) and hamster anti-mouse CD28 (BD Biosciences, 553294) for 72 hours. As described in detail previously [34 (link)] ATP luminescence was employed as a measure of cell viability/proliferation (CellTiter-Glo (CTG) Luminescent Cell Viability Assay (Promega, Madison, WI, USA). Luminescence was read using the Infinite 200 PRO (Tecan, Männedorf, Switzerland) with i-control software (Tecan).

Hydrophobic 96-well plates with 0.45 µm pore size PVDF membranes (EMD Millipore S2EM004M99) were coated with capture antibody according to the manufacturer’s instructions for the Mouse IFN-γ ELISpot assay (R&D Systems SEL485). The stimulating antigen was 1 µg/mL PfCSP (3D7) overlapping 15-mer peptide pool. The positive control for cell stimulation was 1 µg/mL hamster anti-mouse CD3e (BD Biosciences 553057). The negative control was culture media alone. Plates were blocked with complete media for at least 2 h. Mouse splenocytes were plated based on spot counting optimization and ranged from 25,000 to 100,000 cells/well. Plates were incubated for 42 h at 37 °C with 5% carbon dioxide for cell stimulation. Wells were probed with the detection antibody according to the manufacturer’s instructions. Following the detection incubation, plates were developed with the ELISpot Blue Color Module according to the manufacturer’s instructions (R&D Systems SEL002) and allowed to dry completely before analysis. Spot counting was performed using an AID ELISpot Reader (Autoimmune Diagnostika).