Single-cell suspensions from the bladders and spleens of sacrificed mice were obtained as previously described [39 (link),40 (link)]. Briefly, splenocytes were obtained by mechanical dissociation, while bladders were minced and digested step-wise with 0.5 mg/mL thermolysin (Roche, Basel, Switzerland) and 1 mg/mL collagenase/dispase (Roche). IFN-γ ELISPOT assays were performed as described [39 (link)] using Multiscreen-HA 96-well plates (MAHA S4510, Millipore, Billerica, MA, USA), anti-IFN-γ monoclonal antibody (R4-6A2, Beckton Dickinson Pharmingen), biotinylated anti-IFN-γ monoclonal antibody (XMG1.2, Beckton Dickinson Pharmingen) and Streptavidin-AP (Roche). 100,000 splenocytes/well in duplicates were incubated with 0.5 μg/mL of H-2Db restricted Uty246–254 peptide or medium alone (control wells) for 16–24 h. Uty-specific responses were defined as the number of IFN-γ spots/105 cells in the Uty-stimulated wells minus the number of IFN-γ spots/105 cells in the control wells (<3 spots/well). T cell staining was performed as previously described [41 (link)], using PE-conjugated Uty246–254 H-2Db-restricted dextramers (Immudex, København, Denmark) and APC-labeled CD8α (clone 53-6.7, eBioscience, San Diego, CA, USA). Flow-cytometer cell acquisition and analysis were performed as described above.
Multiscreen ha 96 well plates
Manufactured by Merck Group
Sourced in United States
Multiscreen-HA 96-well plates are a laboratory equipment product designed for high-throughput filtration and sample preparation. The plates feature a hydrophilic membrane that facilitates efficient liquid sample separation and retention.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin ap, by Roche (1 mentions)
Collagenase dispase, by Roche (1 mentions)
Thermolysin, by Roche (1 mentions)
Avidin d hrp, by Vector Laboratories (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Porcine ifn γ elispot basic kit, by Mabtech (1 mentions)
Elispot reader, by Mabtech (1 mentions)
Rpmi medium, by Thermo Fisher Scientific (1 mentions)
Immunospot reader, by Cellular Technology (1 mentions)
5 protocols using multiscreen ha 96 well plates
1
Isolation and Analysis of Murine Immune Cells
2
Enzyme-Linked Immunospot Assay for Antibodies
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Millipore Multiscreen-HA 96-well plates (Millipore #MAHA N4510) were coated with anti-mouse IgG, IgA, IgM (Rockland) diluted to 5 μg/mL in PBS and incubated overnight at 4°C. Plates were then washed with PBST (1X PBS, 0.05% Tween 20) and PBS (1X, Life Technologies) (1x PBST, 3x PBS washes) and blocked by 2 hr incubation at 37°C with cIMDM. Media was then replaced with fresh cIMDM, and counted cells from spleens, MLN, and PP were added. Plates were incubated overnight at 37°C and, following washes (4x PBS, 4x PBST), biotin-conjugated anti-mouse IgG and IgA antibodies (Southern Biotech) were added at a concentration of 0.5 μg/mL diluted in PBST 1% FCS and incubated overnight at 4°C. Plates were washed (4x PBST) before incubation with a 1:1000 dilution of HRP avidin D (Vector Laboratories) in supplemented PBS (1X PBS, 0.05% Tween 20, 1% FCS) for 1–3 hr at room temperature. After washes (3x PBST, 3x PBS), AEC substrate (0.3mg 3-amino-9-ethylcarbazole in 0.1 M Na-Acetate buffer, pH 5, 0.3% hydrogen peroxide) was added and color reactions were allowed to proceed for 2–10 minutes before washing with distilled water. Plates were kept in the dark to dry until read and counted with the aid of a CTL ImmunoSpot 5.1.36 analyzer.
3
Quantifying IFN-γ-secreting T-cells by EliSpot
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The number of IFN-γ-secreting T-cells was determined by the Porcine IFN-γ EliSpot BASIC kit (3130-2A, MabTech, OH, USA) using PBMCs and splenocytes pulsed with peptides as previously described (54 (link), 69 (link)). In brief, each sample was assayed in triplicate in MultiScreen-HA 96-well-plates (MAIPS4510, Millipore, MO, USA) with 2.5 x 105 cells/mL cells pulsed with 2.5 μg/mL of each peptide in cRPMI 1640 media. Peptide screening was carried out using four pools [A-D] containing 18 9-mer peptides and a final pool [E] contained the remaining 16 peptides (Table 2 ). Reactive pools were then tested at the individual peptide level at the same concentration indicated earlier. For each test, positive and negative controls were Phytohemagglutinin (PHA) mitogen at a concentration of 5 μg/mL and media alone, respectively. After a 48-h incubation at 37°C in 5% CO2 atmosphere, plates were developed as per the MabTech protocol, the membranes air-dried in the dark, and spots were detected using EliSpot reader (MabTech, OH, USA) and AID software (version 3.4; AutoImmun Diagnostica, Strasburg, Germany). Data are presented as Spot Forming Cells (SFC)/106 PBMCs or splenocytes based upon the mean number of peptide-specific IFN-γ producing cells after subtracting the negative control mean counts as background.
4
Quantifying IgE and IgG1 Secretion
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Cells were incubated at 37 °C for 6–18 h in MultiScreen-HA 96 well plates (Merck Millipore Corporation., Darmstadt, Germany) coated with TNP26-conjugated bovine serum albumin (TNP26-BSA). After removing cells, secreted Abs bound to wells were detected by alkaline phosphatase (AP)-conjugated polyclonal Abs against IgE or IgG1 and NBT-BCIP substrate (Wako). The spot number and the area (pixels) were quantified using Image J software (National Institute of Health, Bethesda, MD).
5
IFN-γ ELISpot Assay for Porcine Immune Response
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IFN-γ ELISpot assays were performed as previously described (21 (link)) with minor modifications. Briefly, MultiScreen-HA 96-well plates (Merck) coated with anti-porcine IFN-γ monoclonal antibody (mAb; clone P2G10; BD Biosciences, San Jose, CA, USA) were blocked in Roswell Park Memorial Institute (RPMI) medium (Thermo Fisher Scientific) supplemented with 10% HI FBS and antibiotics (complete RPMI) for 2 h at 37°C in 5% CO2. Freshly isolated PBMCs and BALCs (Exp 1) or thawed cryopreserved PBMCs (Exp 2) were seeded at a density of 5 × 105 cells/wells in triplicate. Cells were restimulated with H3N2 or PRRSV-2 at a multiplicity of infection (MOI) of 3.1 and 3.3, respectively, in complete RPMI for 18 h at 37°C in 5% CO2. Cells cultured in complete RPMI or with 10 µg/ml of concanavalin A (ConA, Merck) were used as controls. IFN-γ-secreting cells were revealed and enumerated as described, using the biotinylated anti-porcine IFN-g detection mAb (clone P2C11, BD Biosciences) followed by the secondary streptavidin–alkaline phosphatase and BCIP/NBT reagent (21 (link)). Plates were automatically counted using ImmunoSpot Reader (Cellular Technology Limited, Ohio, USA). Results were expressed as the number of IFN-γ-producing cells per 106 cells minus the average number of IFN-γ-producing cells per 106 cells in medium-only stimulated wells.
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