Mouse ifn γ elispotplus kit

Assessment of the IFN-γ T cell response was performed using the Mouse IFN-γ ELISpot^PLUS kit (Mabtech) following the manufacturer’s instructions. Briefly, anti-IFN-γ pre-coated plates were blocked with DMEM + 10% FBS for at least 30 min, then cells were added at 2.5 × 10⁵ cells per well for negative control (media only) and SARS-CoV-2 peptide pools (15-mers overlapping by 11; JPT Peptides) (1 µg mL⁻¹) in 200 µL final volume per well. The positive control wells contained 5 × 10⁴ cells per well in 200 µL final volume per well with 5 µg mL⁻¹ of ConA. Plates were incubated overnight at 5% CO2, 37 °C incubator and developed as per the manufacturer’s protocol. Once dried, plates were read using the AID ELISpot reader ELR03 and AID ELISpot READER software (Autoimmun Diagnostika GmbH).

First, spleens were collected from mice, and splenocytes were prepared into single cell suspensions in 6-well plates (CellPro, Suzhou, China) containing RPMI 1640 medium (Gibco, Grand Island, NY, USA). These suspensions were then added to 15 mL centrifuge tubes, and mouse lymphocyte isolates (Dakewe Biotech Co., Ltd., Shenzhen, China) were obtained according to the instructions for mouse lymphocyte isolates. The mouse IFN-γ ELISPOTPLUS kit (ALP) and mouse IL-4 ELISPOTPLUS kit (ALP) (Mabtech, Nacka Strand, Sweden) were utilized following the manufacturer’s instructions. Approximately 5 × 10⁵ splenic lymphocytes were added to the wells and cultured in a medium with a final concentration of 2 μg/mL S1 protein. Ten microliters of PHA (Dakewe Biotech Co., Ltd., Shenzhen, China) was added to the positive control wells, while the negative control wells contained cells and RIPA 1640 medium only. The plates were incubated in an incubator containing 5% CO₂ at 37 °C for 24 to 36 h. Post incubation, the spots were color developed as per the instructions provided and then read with an ELISpot reader (CTL, Shaker Heights, OH, USA).

In triplicate wells of 96-well plates, pooled splenocytes from GTL001-immunized or placebo-injected mice (1 × 10⁶ cells in 100 μL complete medium) were mixed with 100 μL of complete medium containing 1 μg/mL of major histocompatibility complex (MHC) class I (H-2^b)-restricted peptides (HPV16 E7_49-57, HPV18 E7_AS43-49, or ovalbumin_257-264), no addition (negative control), or 1 μg/ml hamster anti-mouse CD3_Ɛ antibody (BD Biosciences; positive control for polyclonal T-cell responsiveness). After 20 h at 37°C, frequencies of IFN-γ-secreting CD8⁺ T cells were assessed using a mouse IFN-γ ELISpot PLUS kit according to the manufacturer’s instructions (Mabtech, Nacka Strand, Sweden). Spot-forming cells were counted using a Bioreader 5000 Pro S plate reader (Biosys, Karben, Germany).

To detect RBD-specific T cell responses, splenocytes from vaccinated mice were analyzed using the IFN-γ ELISpot assay. Briefly, BALB/c mice were sacrificed at days 7, 14 or 42 post-vaccination, and the spleens were harvested aseptically. The spleens were homogenized, and single-cell suspensions were incubated with 1 × ACK buffer to remove erythrocytes, washed, and resuspended to a cell concentration of 6 × 10⁶ cells. The cells were plated in triplicate and stimulated with 2 µg/ml of Wuhan-Hu-1 or B.1.351 RBD peptide pools (6 pools consisting of 10–11 15mers overlapping with 12 aa) for 24 h. Cells without peptide stimulation were used as negative controls. Stimulated splenocytes were analyzed for IFN-γ response using a mouse IFN-γ ELISpot Plus kit (Mabtech AB, Stockholm, Sweden). Spot-forming units (SFU) were measured using an IRIS ELISpot reader and APEX™ software (Mabtech AB). Results are shown as the mean number of IFN-γ + spots/10⁶ splenocytes with a subtracted background.

Splenocytes were processed and IFN-γ producing cells specific for FLuc or spike were measured using a mouse IFN-γ ELISpot-plus kit (MabTech) as described previously [18 (link)]. Briefly, splenocytes were stimulated with the FLuc peptide pool (1 μg/peptide/mL, 0.4% DMSO), the spike peptide pool (1 μg/peptide/mL, 0.4% DMSO), or 0.4% DMSO in medium (negative control). All samples were run in duplicates. Plates were counted on an AELVIS ELISpot reader, and the numbers of spot-forming units (SFU) per 10⁶ cells were calculated. Background was defined as 95^th percentile of values from the 0.4% DMSO in medium.

Spleens were harvested and dissociated into single cell suspension of splenocytes by manually grinding the spleen over a 40 µm filter (Falcon) and red blood cells lysed using ACK Lysing Buffer (Thermo Fisher). Cells were washed with PBS and resuspended in RPMI 1640 with l-Glutamine (Thermo Fisher Scientific), 10% fetal bovine serum (Thermo Fisher Scientific), and 1% Antibiotic–Antimycotic (Thermo Fisher Scientific). Cytokine analysis was performed using the mouse IFN-γ ELISpotPLUS Kit (Mabtech), according Manufacturer’s protocol. Briefly, plates were blocked with serum-containing culture media and stimuli and cell suspension (400,000 per well) added. B16 antigen pool used was a mixture of GP100 (KVPRNQDWL), TRP-2 (SVYDFFVWL), and TYR (YMDGTMSQV) (AnaSpec), with each having a final assay concentration of 2.5 µg/mL. Plates were wrapped in foil and incubated for 18 h at 37 °C, 5% CO₂. Following stimulation, the cells were removed, plate washed, and 1 µg/mL detection antibody added for 2 h at room temperature. Wash was repeated and 1× Streptavidin-HRP added and incubated for 1 h at room temperature. Finally, plates were washed and TMB substrate added, incubated for 4 min in the dark for spot development, then washed out using tap water. Plates were allowed to dry and counted (ZellNet Consulting).