PBMCs were resuspended in RPMI-1640 (Lonza) with 10 % fetal bovine serum (Hyclone), 100 U/ml penicillin, 0.1 mg/ml streptomycin (Sigma Aldrich) (negative control) or in medium containing 1 µg/ml R848 (MabTech) and 1 µg/ml rhIL-2 (MabTech) for expansion of B-cells. Two million cells were added per well in 24-well plates (Nunc) and incubated for 6 days at 37 °C, 5 % CO2. ELISPOT plates (Millipore) were coated with 15 µg/ml anti-human IgG (MabTech), 10 µg/ml SARS-CoV-2 spike protein, or PBS only (negative control) at 4 °C overnight. Lymphocytes were incubated in ELISPOT plates for 16 h (37 °C, 5 % CO2). IgG+ MBCs were detected with 1 µg/ml biotinylated anti-IgG monoclonal antibody (MabTech) for 2 h at room temperature followed by Streptavidin-HRP (1:1000) (MabTech). Spots were developed with 3,3′,5,5′-tetramethylbenzidine (TMB) ELISPOT substrate (MabTech). The plates were counted using an ELISPOT reader (Advanced Imaging Devices, Germany). Spike-specific spots were calculated as the mean of duplicate wells after subtraction of negative controls and presented as spot forming units per million PMBCs (SFU/106 PBMCs).
Rhil 2
Manufactured by Mabtech
Sourced in Sweden
RhIL-2 is a recombinant human interleukin-2 protein. Interleukin-2 is a cytokine that plays a crucial role in the activation and proliferation of T cells, natural killer cells, and other immune cells. RhIL-2 can be used for in vitro research applications.
Automatically generated - may contain errors
Lab products found in correlation
Elispot plate, by Merck Group (2 mentions)
Anti human igg, by Mabtech (1 mentions)
24 well plate, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Rpmi 1640, by Lonza (1 mentions)
Streptavidin hrp, by Mabtech (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Streptavidin alkaline phosphatase, by Mabtech (1 mentions)
Bcip nbt, by Mabtech (1 mentions)
Mt91 145, by Mabtech (1 mentions)
Mt78 145, by Mabtech (1 mentions)
4 protocols using rhil 2
1
SARS-CoV-2 Spike Protein IgG ELISPOT Assay
2
Isolation and ELISPOT Assay of Memory B Cells
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PBMCs were isolated, washed and checked cell viability by trypan blue exclusion test. PBMCs were adjusted to a concentration of 1 × 106 cell/mL and distributed into 24-well culture plates. The cells were stimulated with a mixture of 1 µg/mL of R848 and 10 ng/mL of rhIL-2 (Mabtech, Stockholm, Sweden). Cells were cultured under 5% CO2 at 37 °C for 3 days.
The 96-well ELISPOT plates (Millipore) were coated with 10 µg/mL of rDBL-TH or 15 µg/mL of mAb MT91/145 in PBS at 4 °C overnight. The wells were blocked with R10 (10% FBS in RPMI 1640 media) at RT for 1 hour. Stimulated PBMCs were harvested, washed and incubated in DBL-TH antigen-coated ELISpot wells for 16–24 hours. The wells were washed and incubated with 100 µL of 1 µg/mL mAb MT78/145 at RT for 2 hours. After washing with PBS, 100 µL of Streptavidin-conjugated HRP was added and incubated at RT for 1 hour. The plate was washed and 100 µL of TMB substrate solution was used to detect the spot. After 10 mins, the enzymatic reaction is stopped by rinsing the plate with deionized water. Spots were count by Bioreader 5000 Pro-F gamma ELISPOT Reader (BioSys GmbH, Germany) (Supplementary Fig.3 ). The positive response of antigen-specific MBCs was defined as spot-forming cells (SFC) with 2-fold higher total number of spots relative to the negative control.
The 96-well ELISPOT plates (Millipore) were coated with 10 µg/mL of rDBL-TH or 15 µg/mL of mAb MT91/145 in PBS at 4 °C overnight. The wells were blocked with R10 (10% FBS in RPMI 1640 media) at RT for 1 hour. Stimulated PBMCs were harvested, washed and incubated in DBL-TH antigen-coated ELISpot wells for 16–24 hours. The wells were washed and incubated with 100 µL of 1 µg/mL mAb MT78/145 at RT for 2 hours. After washing with PBS, 100 µL of Streptavidin-conjugated HRP was added and incubated at RT for 1 hour. The plate was washed and 100 µL of TMB substrate solution was used to detect the spot. After 10 mins, the enzymatic reaction is stopped by rinsing the plate with deionized water. Spots were count by Bioreader 5000 Pro-F gamma ELISPOT Reader (BioSys GmbH, Germany) (Supplementary Fig.
3
Immortalization and Stimulation of hPBMCs
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hPBMCs, purchased from different commercial sources (Lonza, Basel, Switzerland; StemCell Technologies, Vancouver, BC, Canada; iXCells Biotechnologies, San Diego, CA, USA; Zenbio, Inc., Durham, NC, USA), were maintained in a Roswell Park Memorial Institute 1640 (Gibco, Grand Island, NY, USA) medium, supplemented with 10% (v/v) fetal bovine serum (FBS, Gibco) and 1% (v/v) penicillin/streptomycin (Gibco) at 37 °C, with 5% CO2 and 95% humidity. For the immortalization of hPBMCs, the cells were infected with 250 μL of EBV (ATCC, Manassas, VA, USA) per 1 × 108 hPBMCs. Throughout this study, the stimulation of hPBMCs was performed using a combination of 1 μg/mL R848 and 10 ng/mL rhIL-2 (Mabtech, Stockholm, Sweden). Cell counting of hPBMCs was performed using the ADAM-CellT instrument (NanoEntek inc., Seoul, Republic of Korea).
4
IgG Monoclonal Antibody Detection
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Anti-human IgG monocloning antibodies (MT91/145, Mabtech) were diluted with PBS (PH7.4) and coated onto a 96-well polyvinylidene fluoride plate (Millipore) treated with 70% ethanol for 2 min at a concentration of 15 µg/mL (100 µL/well) overnight at 4°C. The plates were washed with PBS for five times (300 µL/well) and blocked using 10% bovine serum (200 µL/well) at room temperature for 1 hour. MB cells were pre-stimulated with a mixture of R848 at 1 µg/mL and rhIL-2 at 10 ng/mL (Mabtech) for 24 hours. Activated MB cells and CD8+ T cells were inoculated into the plates at a ratio of 1:4 and cultured at 37°C and 5% CO2 for 48 hours. After washing the plate five times with PBS (300 µL/well), 100 µL (1 µg/mL) of biotin-anti human IgG mAb (MT78/145, Mabtech) was added to the plates and incubated at RT for 2 hours. After another round of washing, 100 µL streptavidin-Alkaline phosphatase (1:1000, Mabtech) was added and incubated for 1 hour at RT. 100 µL substrate solution (BCIP/NBT, Mabtech) were added and developed until the distinct spots emerge. The plates were analysed using a CTL reader (ImmunoSpot, Cleveland, Ohio, USA).
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