Mouse ifn γ elispot kit

The spleen was isolated under sterile conditions, and the splenic lymphocytes were divided into lymphocyte suspensions according to the instructions of the lymphocyte separation solution (Dakewe Biotech, Beijing, China). For the flow cytometry analysis, the lymphocytes were stained with CD3 (BD, Franklin Lakes, NJ, USA) and detected by flow cytometry (LAR Fortessa, BD, USA). Flow JO software was used to analyze the total T cells populations of the lymphocytes. For the ELISPOT experiment, a mouse IFN-γ ELISPOT kit (MABTECH Inc., Cincinnati, OH, USA) was used according to the manufacturer’s protocol. Briefly, the plate was conditioned and seeded with splenic lymphocytes prior to the addition of the stimulant (95% pure peptides: gB498-505: SSIEFARL) (Sangon Biotech, Shanghai, China), which mainly target CD8⁺ T lymphocytes. The plates were then incubated at 37 °C for 12–48 h. After the incubation step, the cells and medium were removed, and the plate was developed. The colored spots were counted using an automated ELISPOT reader (CTL, Cleveland, OH, USA), with spot-forming cells (SFCs) representing HSV-2-specific IFN-γ-producing T cells [14 (link)].

An ELISpot assay was used to determine the number of F protein-specific IFN-γ-secreting T cells in the spleen. Splenocytes were stimulated overnight with a peptide pool consisting of 15-mer peptides with an 11-amino-acid-overlap, spanning the whole sequence of the F protein of RSV A2. The numbers of spot-forming units (SFU) were determined using mouse IFN-γ ELISpot kit (MabTech), according to the manufacturer’s instruction, and calculated to numbers of SFU per 10⁶ cells. Background levels were calculated as the 95% percentile of the SFU observed in non-stimulated splenocytes.

An ELISpot assay for the detection of IFN-γ-secreting mouse splenocytes was performed with a mouse IFN-γ ELISpot kit (Mabtech). Feshly harvested splenocytes of 5 × 10⁵ per well incubated with pools of NiV G peptides (18-mers with 10 amino acid overlap). The final concentration of each peptide was 2 μg/ml. The cells were then incubated with 5% CO2 at 37 °C. 24 h later, IFN-γ spot-forming cells were detected by staining membranes with anti-mouse IFN-γ-biotin (1 μg/ml) followed by streptavidin-ALP. Phorbol 12-myristate 13-acetate and 10 μg/ml ionomycin (Dakewe) was added to the positive-control group, whereas the negative-control group received no stimuli. Analysis was performed using the CTL ImmunoSpot Analyzer and ImmunoSpot Software (Cellular Technology). Spot-forming unit (SFU) per million cells was calculated.

ELISpot assays were performed with the Mouse IFN-γ ELISPOT Kit (Mabtech, Cincinnati, OH, USA) following the manufacturer’s instructions. Briefly, peripheral blood mononuclear cells (PBMCs) were isolated from mice’s spleens using a lymphocyte isolation technique (Ficoll-Paque PREMIUM; GE Healthcare, Piscataway, NJ, USA) and plated in duplicate wells. Recombinant Spike proteins of wild-type and variant strains of SARS-CoV-2 (Sino Biological, Inc., Beijing, China) were added to separate wells as stimulators. Phytohemagglutinin (PHA) was added as the positive control. The plates were incubated at 37 °C for 24 h, after which cells and medium were removed to allow spot development. An automated ELISPOT reader (CTL, Cleveland, OH, USA) was used to count the colored spots. Spot-forming cells (SFCs) were T cells that produced SARS-CoV-2-specific IFN-γ.

Mouse splenocytes were harvested for analysis with IFN-γ ELISpot as previously described [43 (link)], using restimulation with 2 µg/mL of a pool of 275 peptides. These peptides are synthesised as 15 mers and overlapping by 10 amino acids, corresponding to MERS-CoV spike protein, and have been previously tested for mouse and human samples [25 (link),30 (link),31 (link)]. Each sample of splenocytes was plated in duplicate wells at three different numbers of cells: 500,000, 250,000, and 125,000 cells per well; thus, each sample was plated in a total of six wells. Cells were incubated with the peptide for 18–20 h at 37 °C, plates were washed, and spots were developed using a Mouse IFN-γ ELISpot kit (MabTech, Nacka Strand, Sweden), following the manufacturer’s instructions. The total number of spots in each well was counted using an AID ELISpot reader (AID Autoimmun Diagnostika, Straßberg, Germany). The numbers of spots were calculated to report the spots per 1 million splenocytes for each well. Further duplicate wells were plated with 250,000 cells from each sample and left without peptide restimulation. In the absence the restimulation, the frequencies of IFN-γ⁺ cells were subtracted from the cellular frequencies of the tested restimulated samples. IFN-γ secreting splenocytes were reported as the average of spot forming cells (SFCs) per million splenocytes for each sample.

The relative numbers of IFN-γ expressing T cells in single cell spleen suspensions were measured using the mouse IFN-γ ELISPOT kit (#88-7314-88, MAB TECH, Sweden) as per the manufacturer’s instructions. Briefly, splenocytes (2 × 10⁵, n = 4) were added to each well of pre-coated 96 well plate in triplicate, and stimulated with or without 100 μl/well (10 μg/ml) of a purified HA protein at 37°C for 40 h. After incubation, the cells were removed and the plate was washed and incubated with 100 μl/well (1 μg/ml) of a biotinylated detection antibody (#R4-6A2-biotin) for 2 h at room temperature. Then the plate was washed and incubated with 100 μl/well of Streptavidin-HRP (1:1000) for 1 h at room temperature. Finally, the plate was treated with 100 μl/well of the ready to use TMB substrate solution and incubated until distinct spots emerged, and the reaction was stopped by washing extensively in deionized water. The number of spots was counted in a dissection microscope and the results were expressed as spot forming cells (SFC) per million cells.