Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved, as previously described.38 (link) We analyzed PBMCs that had received prior in vitro stimulation with the TGF-β-15 epitope, as previously described.35 (link) After incubation in 9–10 days, the cells were counted using a Countess II Automated Cell Counter (Thermo Fisher) and the occurrence of T cells specific to TGF-β-15 was assessed with an interferon-ɣ (IFN-ɣ) enzyme-linked immunospot (ELISpot) assay. Cells were plated out in triplicates with a concentration of 200,000 cells per well and stimulated with peptide for a final concentration of 5 µM in the well. PVDF membrane plates (Merck, Germany) coated with primary IFN-ɣ specific antibody (Mabtech, Sweden) were used for the ELISpot assays. After incubation overnight the cells were poured off, and following manufacturer’s protocol (Mabtech, Sweden) the wells were coated with secondary antibody, streptavidin-alkaline phosphatase (ALP), and enzyme substrate with washing of wells with phosphate buffered saline before and between each step. The plates were counted using the ImmunoSpot S6 Ultimate Analyzer (CTL Analyzers, Shaker Heights, Ohio, USA) when dry. The normalized mean spots were defined as the mean number of spots in peptide-stimulated wells subtracted by the mean number of spots in negative control wells.
Pvdf membrane plates
Manufactured by Merck Group
Sourced in United States
PVDF membrane plates are a type of lab equipment used for a variety of filtration and separation applications. The PVDF (polyvinylidene fluoride) membrane provides a durable and chemically-resistant surface for various sample processing tasks. The core function of these plates is to facilitate efficient liquid filtration and separation across a range of scientific and industrial settings.
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Lab products found in correlation
Immunospot reader, by Cellular Technology (2 mentions)
Vector blue alkaline phosphatase substrate kit 3, by Vector Laboratories (2 mentions)
Anti human igg, by Jackson ImmunoResearch (2 mentions)
Alkaline phosphatase conjugated anti human igg, by Jackson ImmunoResearch (2 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (2 mentions)
Rpmi 1640, by Corning (2 mentions)
Countess 2 automated cell counter, by Thermo Fisher Scientific (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by LGC (1 mentions)
Aec substrate, by BD (1 mentions)
Mouse cd4 t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Minimum non essential amino acids, by Thermo Fisher Scientific (1 mentions)
Stable diaminobenzidine dab, by Thermo Fisher Scientific (1 mentions)
β mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Mabtech (1 mentions)
Immunospot 3, by Cellular Technology (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Elispot assay kit, by BD (1 mentions)
Aec substrate, by Vector Laboratories (1 mentions)
12 protocols using pvdf membrane plates
1
Quantifying Antigen-Specific T Cell Responses
2
SIV-specific IFN-γ ELISpot Assay
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The day before the assay, PVDF-membrane plates (Merck Millipore, Burlington, MA, USA) were coated with 50 µL of 5 µg/mL monoclonal antibody anti-IFN-γ (clone 1-D1K or GZ-4, Mabtech, Mariemont, OH, USA), and incubated overnight at 4 °C. Plates were washed 4 times with complete RPMI and then incubated with fresh complete RPMI for a minimum of 30 min at 37 °C. Two hundred thousand cells were added to the wells and stimulated with 2 µg/mL of peptide pools spanning SIV-Gag, HIV-Env C-terminal or SIV-Nef (AIDS Reagent Repository, NIH-NIAID catalog numbers 6204, 6451, and 8762, respectively). Medium only or PMA-Ionomycin were used as negative and positive controls, respectively, and incubated for 20 h at 37 °C. Plates were washed 4 times with PBS + 0.05% Tween-20 and captured IFN-γ spot-forming units were revealed using anti-IFN-γ secondary antibody coupled with biotin, then incubated with Avidin-D-HRP. Spots were revealed using TMB (SeraCare, Milford, MA, USA), washed with tap water and counted with a CTL-6 Immunospot reader (Cellular Technology Limited CTL, Shaker Heights, OH, USA) after drying the plates.
3
Influenza-specific Antibody-Secreting Cells
4
PVDF-based ELISpot for CHIKV-specific T cells
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Polyvinylidene difluoride (PVDF) membrane plates (Millipore) were humidified with 35% ethanol and washed with water. Wells were coated with anti-gamma interferon (IFN-γ) capture antibody (clone AN18; Mabtech) overnight at 4°C. Mice were sacrificed at 6 dpi, and joint footpad and pLN cells were isolated as described above. Splenocytes, joint footpad cells, and pLN cells were added at 2 × 105, 2.5 × 104, and 5 × 104 cells per well, respectively. Ex vivo CD4+ enrichment was performed using a mouse CD4+ T cell isolation kit II (Miltenyi Biotec) according to the manufacturer's instructions. Stimulation of CHIKV-specific T cells was done in complete RPMI containing 30 U/ml IL-2 and 1.5 × 106 VeroE6-derived SGP011 virions per well. Complete RPMI containing 30 U/ml IL-2 was included as a negative control. For all wells containing joint footpad cells, 1.5 × 105 splenocytes from NI mice were added to serve as antigen-presenting cells (APCs). The plates were incubated at 37°C and 5% CO2 for 18 h. After incubation, cells were removed and wells were washed six times using PBS. Spot detection was done using a mouse IFN-γ enzyme-linked immunospot (ELISpot) kit with alkaline phosphatase (ALP) (Mabtech) following the manufacturer's instructions.
5
Vaccine-Induced Antibody-Secreting Cell Responses
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Peripheral blood mononuclear cells (PBMCs) were collected from study persons prior to (days 0 and 28) and 7 days after each vaccine dose (days 7 and 35) for measurement of ASCs. Cryopreserved PBMCS were used for determining the number of ASCs by enzyme-linked immunosorbent spot (ELISPOT) assays as described previously (18 (link)). Briefly, 96-well polyvinylidene difluoride (PVDF) membrane plates (Millipore) were coated with 1 μg per well of GI.1 or GII.4 (consensus) VLPs or phosphate-buffered saline (PBS) overnight at 4°C. Cryopreserved PBMCs were thawed, and doubling dilutions were prepared in AIM-V medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen), sodium pyruvate (Invitrogen), minimum nonessential amino acids (Invitrogen), and β-mercaptoethanol (Invitrogen), starting with 5 × 105 PBMCs per well in the first row. Following incubation in a humidified incubator with 5% CO2 at 37°C for about 18 h, IgA and IgG ASCs were detected using goat anti-human IgA and IgG antibodies conjugated to horseradish peroxidase (HRP), respectively (Southern Biotech), and stable diaminobenzidine (DAB) as a substrate (Invitrogen). The membrane was allowed to dry overnight, and spots were imaged using an ImmunoSpot reader (Cellular Technology Limited).
6
IgM and IgG ELISpot Assay for B Cell Analysis
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IgM and IgG enzyme-linked immunosorbent spot (ELISpot) assays (MabTech, Nacka Strand, Sweden) were performed following the instructions of the manufacturers. Briefly, 96-well polyvinylidene difluoride (PVDF) membrane plates (Millipore, Burlington, MA, USA) were coated with mouse anti-human IgM and IgG monoclonal antibodies (MabTech). Meanwhile, 3.2 × 105 resting B cells/well were seeded in 96-well round bottom microplates and incubated in the presence of the preformed complexes and 50 ng/ml IL-2, IL-6, and IL-10 (ImmunoTools, Friesoythe, Germany) at 37°C and 5% CO2 for 72 h. Then, 5 × 104 differently treated cells were placed into the wells of the PVDF plates and incubated at 37°C and 5% CO2 for 20 h. Then, biotinylated mouse anti-human IgM and IgG (MabTech, Nacka Strand, Sweden), streptavidin-conjugated HRP (MabTech), and TMB (MabTech) were added to the samples. B cells secreting IgM and IgG were assessed by an ImmunoSpot Reader (Cellular Technology, Shaker Heights, OH, USA) using ImmunoSpot 3 software (Cellular Technology). Images were prepared with PaintShop Pro.
7
ELISPOT for IFNγ Production
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IFNγ specific ELISPOT was performed according to the manufacturer's instructions (Mabtech). 200,000 splenocytes per well were plated out on PVDF membrane plates (Millipore) followed by stimulation for 19 hours in 37°C with 2 μg/ml SIINFEKL, 2 μg/ml OVA323-339 peptide (Innovagen), 2 μg/ml Concanavalin A (Sigma), 2 μg/ml αGC, or culture media as a control.
8
Cytotoxic T-Cell Activation ELISpot
9
ELISpot Assay for CTL Response
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96-well PVDF-membrane plates (Millipore) were coated with IFN-gamma capture antibody overnight at 4°C. On the day of the assay, the plates were blocked for 2 hours in RPMI-1640 complete medium and washed prior to use in the ELISpot assay. In vitro generated CTLs were assayed 7 days after the final restimulation. Peptide specific CTLs were harvested, counted, and cultured overnight with appropriate antigen presenting cells that were unpulsed or pulsed with synthetic peptides (T2 or HepG2 cells) or antigen presenting cells that are productively infected with HBV (HepDE19, HepG2.2.15, and 293/T/A2 Ad-HBV infected cells). The next day the assay was developed according to the manufacturer's instructions (BD Biosciences, San Jose, CA). In vivo generated CTLs were assayed 7 days after the final peptide injection. Spleens were harvested and homogenized into a single cell suspension. After lysis of RBCs, the cells were extensively washed, counted, and cultured overnight with HepG2 cells unpulsed or pulsed with synthetic peptides or HepDE19 cells. The next day the assay was developed according to the manufacturer's instructions (BD Biosciences). For both ELISpot assays, spots were quantified using the ELISpot Reader System (AID, San Diego, CA).
10
ELISpot Assay for HA-Specific ASCs
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HA-specific antibody-secreting cells (ASCs) were detected using an ELISpot protocol as previously described with modifications.19 (link) Briefly, cultures that were either stimulated with influenza antigens for 7 days or left unstimulated were resuspended and enumerated, then plated on HA-coated (see Table S5 ) and blocked 96-well PVDF membrane plates (Millipore). Each sample was plated in duplicate and total live-cell counts ranged from 3.6 x 104 to 1.08 x 105 cells per well. Cells were incubated on these membranes, undisturbed for 5h at 37°C. Plates were then washed and treated with horseradish peroxidase-conjugated anti-IgG/IgA/IgM secondary antibody (Abcam). After incubation overnight at 4°C, plates were washed and developed with AEC substrate (Vector), washed 20 times with water, dried, and spots were enumerated. The frequency of ASCs out of total B cells was determined from B cell flow cytometry data analysis and the direct cell enumeration counts.
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