The BD ELISPOT kit (BD Biosciences) was used according to the manufacturer's instructions. Noncultured PBMCs (5 × 105 per well) were incubated with peptide antigens (10 μg/mL) for 20 hours at 37°C and 5% CO2. The following HSP105 antigens were used: HLA‐A2‐restricted A2‐7 (RLMNDMTAV) or A2‐12 (KLMSSNSTDL) for HLA‐A2‐positive PBMCs and HLA‐A24‐restricted A24‐1 (NYGIYKQDL) or A24‐7 (EYVYEFRDKL) for HLA‐A24‐positive PBMCs. The PBMCs incubated with HLA‐A2‐restricted HIV19‐27 (TLNAWVKVV) or HLA‐A*24:02‐restricted HIV583‐591 (RYLKDQQLL) peptides (ProImmune) were used as negative control. The number of CTL spots was calculated automatically by the Eliphoto system (Minerva Tech). To eliminate nonspecific immune responses unrelated to HSP105 peptides, the number of spots produced by HSP105‐specific CTLs was determined by subtracting the number of spots generated against HLA‐matched HIV peptides from that against HSP105 peptides; if HIV peptides produced more than 100 spots and the difference between HSP105 and HIV peptides was less than 30 spots, the results were excluded. All analyses were carried out in duplicate.
Elispot kit
Manufactured by BD
Sourced in United States
The ELISPOT kit is a laboratory equipment used to detect and quantify the production of specific proteins, such as cytokines, by individual cells. It provides a sensitive and reliable method for analyzing the functional activity of cells without the need for cell culture. The kit contains all the necessary components, including pre-coated plates, detection antibodies, and reagents, to perform the ELISPOT assay.
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Lab products found in correlation
Streptavidin hrp, by BD (2 mentions)
96 well filtration plate, by Merck Group (2 mentions)
2 4 6 trinitrobenzenesulfonic acid, by Merck Group (2 mentions)
Goat anti mouse igg hrp, by Southern Biotech (2 mentions)
5 and 6 carboxyfluorescein diacetate succinimidyl ester cfse, by Thermo Fisher Scientific (2 mentions)
Goat anti mouse igm hrp, by Southern Biotech (2 mentions)
Magnetic anti goat mouse ig microbeads, by Miltenyi Biotec (2 mentions)
Rgg and bsa, by Merck Group (2 mentions)
Whole chicken ovalbumin ova, by Merck Group (2 mentions)
Rylkdqqll, by ProImmune (1 mentions)
Ovalbumin (ova), by Merck Group (1 mentions)
Anti cd8α microbeads, by Miltenyi Biotec (1 mentions)
Hypotonic lysis buffer, by BD (1 mentions)
Cd3e percp cy5, by Thermo Fisher Scientific (1 mentions)
Streptavidin horseradish peroxidase hrp, by Merck Group (1 mentions)
Immunospot series 5 analyzer, by Cellular Technology (1 mentions)
Aec substrate reagent, by BD (1 mentions)
Anti mouse ifn γ, by BD (1 mentions)
Aec substrate, by BD (1 mentions)
Biotinylated anti mouse ifn γ, by BD (1 mentions)
26 protocols using elispot kit
1
ELISPOT Assay for HSP105-Specific CTLs
2
Measuring Antigen-specific CTL Response
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An ex vivo IFN‐γ ELISPOT assay was carried out to measure the antigen‐specific CTL response, as described previously.30 Briefly, a peripheral blood sample (10 mL) was obtained from each patient before the first vaccination and every month after the first vaccination, and centrifuged with a Ficoll‐Paque gradient. The PBMCs obtained from enrolled patients were frozen before immunologic analysis. All PBMCs were incubated in the same plate and simultaneously analyzed by ex vivo IFN‐γ ELISPOT assay. Noncultured PBMCs (5 × 105 per well) were added to each plate containing 1 type of the peptide antigen (10 μg/mL) and incubated for 20 hours at 37°C in 5% CO2. The peptide antigens used in this assay were HLA‐A*24:02‐restricted KOC1 peptide (S‐488403), HLA‐A*24:02‐restricted FOXM1 peptide (OTSGC‐A24‐Fo), and HLA‐A*24:02‐restricted KIF20A peptide (OCV‐105); each of these was included in each plate. The PBMCs plus HLA‐A*24:02‐restricted HIV peptide (RYLKDQQLL; ProImmune) were used as the negative control. The number of spots was automatically calculated by the Eliphoto system (Minerva Tech) using the BD ELISPOT kit (BD Biosciences). These assays were carried out in duplicate. Each peptide‐specific spot number showed the number of each peptide‐specific spot counted by subtracting the spot number in a well of HIV peptide.
3
In Vitro Cytotoxicity Assay Protocol
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IFN-γ ELISpot assays were performed using the ELISpot kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the instructions provided by the manufacturer, and in vitro cytotoxicity assays were performed based on previous studies [33 (link)]. Simply put, Lewis cells were used as target cells and labeled with antigen peptides (final concentration: 5 μg/mL) or without antigen peptides and incubated for 2 h. Target cells incubated with peptides were stained with high concentrations (5 μM) of CFSE, while non-peptide-labeled target cells were stained with low concentrations (0.5 μM) of CFSE. Target cells stained with high and low concentrations of CFSE were evenly mixed 1:1 (a total of 1 × 105 target cells), and then different numbers of spleen cells (effector cells) from vaccinated mice were mixed with target cells in different ratios. Effector cells and target cells were incubated at 37 °C for 8 h, and cytotoxicity was analyzed by flow cytometry, which detects a decrease in the percentage of specific target cells.
4
Antigen-Specific Splenocyte Cytotoxicity
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IFN-γ molecules released by antigen-stimulated splenocytes were detected using an ELISpot kit ((BD Biosciences) as previously described.41 (link)Cytotoxicity assays were performed in accordance with previously published procedures.20 (link) In brief, MSf+ Lewis and Lewis cells as target cells were labeled with different concentrations of CFSE fluorescent dye. A total of 5 × 105 splenocytes isolated from immunized mice were incubated at different effector-to-target (E:T) ratios with the target cells for 6–8 h at 37 C and 5% CO2. The cytotoxicity was analyzed using flow cytometry, and the antigen-specific lethality was calculated according to the formula: specific cytotoxicity = [1 − (MSf+ Lewis cells/unloaded cells from the immunized group)/(Lewis cells/unloaded cells from the naïve group)] × 100%.
5
Evaluating Antigen-Specific T Cell Responses
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Day 8 BMDCs were seeded at a density of 2 × 104 cells in 96-well plate, with or without treatment using 500 μg/ml of OVA (Ovalbumin, Sigma, A5503) and tumor antigen (ECM + lysate). After 24 h, treatment media were removed and cells were washed with PBS for 3 times. 1 × 105 MACS-purified OT-I T cells from OT-1 mice in 200 μl of RPMI 1640 complete medium were added and co-cultured with DCs (DC:T cell = 1:5) for 24 h, then 1 × 104 T cells were transferred into IFNγ mAb pre-coated ELISpot plate for another 24 h. IFNγ producing T cells were detected using ELISpot kit according to the manufacturer’s instructions (BD Biosciences, 551083). Some OVA and tumor antigen treated DCs and DC-cocultured T cells were analyzed by flow cytometry.
6
Quantification of Antigen-Specific Cytokine-Secreting Cells
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IFN-γ and IL-4-secreting antigen-specific cells were quantified using an ELISPOT kit (BD, CA, USA), as described previously [10 (link)]. Splenocytes (5×105) were added to multiscreen 96-well filtration plates pre-coated with the anti-mouse IFN-γ or IL-4 capture antibody with 10 μg/ml of corresponding antigens or 5 μg/ml of Con A (positive control) in triplicate wells. Notably, the splenocytes of the mice that received the conjugated vaccine were stimulated with PD and HBsAg respectively. The spots were counted using an automated ELISPOT reader. A response was considered positive if the number of spot-forming cells (SFC) per 5×105 splenocytes was greater than 82 (IL-4) or 43 (IFN-γ). SFC values are presented as means ± standard deviation.
7
Antigen-Specific Cytotoxicity Assay
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The IFN-γ ELISpot assay was performed with ELISpot kit (BD Biosciences, San Jose, CA, USA), and in vitro cytotoxicity assays were conducted in accordance with previous studies.48 (link),49 (link) In the antigen-specific ELISpot assay, mouse FAPα and survivin peptide pools (FAPα peptide pools: YVYQNNIYL, YSYTATYYI, HLYTHMTHF, FAVNWITYL, IYSERFMGL, and SSWEYYASI; survivin peptide pools: ATFKNWPFL, IATFKNWPF, QCFFCFKEL, and LTVSEFLKL) were chosen as the stimulators, with the unrelated MUC1 peptide pool (GVTSAPDTR, SAPDTRPAP, DTRPAPGST, PAPGSTAPP, and TRPAPGSTA) as the control. To detect antigen-specific CTLs, Panc02 cells were incubated with FAPα, survivin, or an unrelated MUC1 peptide (5 μg/mL) for 2 h at 37°C. Then, FAPα or survivin-peptide-loaded Panc02 cells were labeled with 5 μM CFSE (CFSE-high cells) for 10 min, while the unrelated-MUC1-peptide-loaded Panc02 cells were labeled only with 0.5 μM CFSE (CFSE-low cells). CFSE-high- and -low-labeled cells were mixed together at a 1:1 ratio. Different numbers of splenocytes from vaccinated mice were then incubated with 5 × 104 of the peptide-loaded Panc02 cells for 8 h at 37 C (E:T ratios were 50:1, 25:1, and 12.5). Specific killing was detected by flow cytometry as the decrease in the percentage of specific targets.
8
Ovalbumin-Specific CD4 T Cell Assay
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The frequencies of IFN-γ-secreting ovalbumin-specific CD4 T cells in the spleens of wild-type (WT) and CIITATgPIV−/− mice were measured using an ELISPOT kit (BD Biosciences; 552569). The WT and CIITATgPIV−/− mice were primarily immunized by subcutaneous injection with 100 μg of ovalbumin emulsified in complete Freund's adjuvant. Two and 4 weeks after the primary injection, second and third injections were performed with 100 μg of ovalbumin in incomplete Freund's adjuvant. One week after the last challenge, 1.0 × 105 CD4+ T cells isolated from the spleens of the immunized mice were cultured with 2.0 × 105 T-cell-depleted WT splenocytes pulsed with ovalbumin (100 μg ml−1) in complete RPMI 1640 media for 20 h at 37 °C in a 5% CO2 incubator. As a positive control, WT CD4 T cells were cultured with T-cell-depleted splenocytes in the presence of anti-CD3 Ab (2C11, 1 μg ml−1), and as a negative control, CD4 T cells from WT or CIITATgPIV−/− mice were cultured with T-cell-depleted splenocytes and without any antigen. The experimental procedures were performed exactly according to the manufacturer's instructions. The resulting spots were counted using a computer-assisted ELISPOT Reader System (AID, Strassberg, Germany).
9
Cytokine Profiling of Immunized Mice
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One week after each immunization, four mice from each group were sacrificed. The spleens were ground through a sterile steel mesh into lymphocyte separation medium. After being centrifuged, the spleen cells were resuspended in complete RPMI-1640 containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin and adjusted to 1×107 cells/ml. For in vitro stimulation, a total of 1×106 splenocytes were incubated with 2 μg of rTs-ES-1 in 200 μl of complete RPMI-1640 in 96-well flat-bottomed cell culture plates for ELISPOT. After cell stimulation for 48 h at 37°C in a humidified atmosphere containing 5% CO2, the cytokines IFN-γ, IL-2, IL-4 and IL-5 were detected using an ELISPOT kit (BD, USA), according to the manufacturer's instructions.
10
Assessing GPC3-specific CD8+ T Cell Response
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Spleens were isolated 17 days after inoculation and mashed using 70 μm filters. Splenocytes were harvested after depletion of RBCs by the hypotonic lysis buffer (BD Biosciences). CD8α-positive splenocytes were isolated by positive selection with anti-CD8α microbeads (Miltenyi Biotec) according to the manufacturer's protocol. The purity of CD8α+ T cells were analyzed with a combination of antibodies specific for CD8α-FITC and CD3e-PerCP-cy5.5 (all purchased from eBioscience, San Diego, CA, USA) by flow cytometry. Then, 2×106 CD8α-positive splenocytes were co-cultured with prepared 5×105 BM-DCs pulsed with 30 μg/ml murine GPC3 proteins (Sino Biological, Inc.) for restimulation. Seven days later, the suspensions containing 4×105 CD8α+ T cells in T-cell medium were added to each well in ELISPOT 96-well plates and stimulated in triplicate with 30 μg/ml GPC3 protein at 37°C for 24 h. The detection of GPC3-specific T cells producing IFN-γ was performed using an ELISPOT kit (BD Biosciences) according to the manufacturer's protocol. Lastly, the plates were dried at normal temperature and the spots were counted with an ELISPOT Reader (CTL Limited) by capturing the images of individual wells. Spot-forming cells were defined as the average number of spots per 4×105 CD8α+ T cells from triplicate wells.
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