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ELISPOT plate reader

Manufactured by Autoimmun Diagnostika
Sourced in Germany

The ELISPOT plate reader is a laboratory instrument designed to detect and quantify the presence of specific immune cells that produce cytokines or other secreted proteins. It is used to perform ELISPOT assays, which are a type of enzyme-linked immunospot assay. The device reads and analyzes the results of the ELISPOT assay, providing data on the number and distribution of the target cells within the sample.

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4 protocols using ELISPOT plate reader

To assess for colony formation in both treated and untreated conditions, 100 cells of each clone were plated in triplicate in a 6-well plate. After a 24 h period, cells were treated with 5 µM of oxaliplatin, 5 µM of 5-fluorouracil or the combination of both drugs. Drug washout was performed 72 h post-treatment. Cells were then incubated for 12–14 days and stained with 0.5% crystal violet (Sigma-Aldrich). Colonies were both manually counted and imaged on the EliSpot plate reader (Autoimmun diagnostika GmbH, Strassberg, Germany). Digital images were quantified by ImageJ (Image Processing and Analysis in Java; National Institutes of Health, Bethesda, MD, USA; http://imagej.nih.gov/).
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Splenocytes or isolated T cells were cultured in RPMI in ELISPOT plates (1 × 106/well; Millipore, Danvers, MA) coated with IFN-γ capture antibody (clone R4.6A2; BD Biosciences) or IL-2 capture antibody (clone JES6-1A12: BioLegend). The samples were stimulated with 20 µg ml−1 of M. tuberculosis whole-cell lysate or subcellular fractions or specifically with a lipoprotein-enriched cell wall fraction, and plates were incubated at 37°C for 24 h. After removal of cells, the plates were washed with PBS followed by PBS with 0.05% Tween 20 (PBST). Biotinylated anti-IFN-γ detection antibody (clone 4S.B3; BD Biosciences) and biotinylated anti-IL-2 detection antibody (clone JES6-5H4; BioLegend) were added for 2 h at 37°C, followed by washing with PBST. Streptavidin-alkaline phosphatase (Sigma-Aldrich) was added to the plates for 1 h (37°C), followed by washing and addition of 5-bromo-4-chloro-3-indolylphosphate–nitroblue tetrazolium (BCIP-NBT) substrate (Sigma-Aldrich). The reaction was stopped by washing the wells with water, and spots were counted using an ELISPOT plate reader (Autoimmun Diagnostika, Strasberg, Germany).
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Multiscreen 96 well filter plates (Millipore, Billerica, MA) were coated with poly-L-lysine followed by dsDNA, histone, or Sm/RNP (ImmunoVision, Springdale, AR) overnight at 4°C, washed once, and blocked with 200 μl of RPMI complete medium with 10% FCS incubated at RT for 2 hours. The blocking solution was discarded, and spleen cells were added at a density of 2×105 per well and incubated at 37°C/5% CO2 for 72 hours. The cells were washed twice with 200 μl per well of deionized water and three times with the wash buffer (PBS containing 0.05% Tween-20). An alkaline phosphatase conjugated anti-mouse IgG secondary antibody was added and incubated for 2 hours at RT. The plates were washed again 4 times with wash buffer and two times with PBS. Substrate solution was added (BCIP-p-toluidine 1 μg/ml in AMP buffer) and incubated in the dark at RT for 5-10 minutes while monitoring spot development. The reaction was stopped by washing the wells with deionized water. The plates were then air dried at RT overnight in the dark, and the spots were quantified by an ELISPOT plate reader (Autoimmun Diagnostika, Strassberg, Germany).
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IFNg secretion was measured by enzyme-linked immunospot assay (ELISpot; R&D Systems). T cells previously expanded against neoantigen peptides were loaded onto ELISpot plates at 25,000 cells per well and stimulated with a T2 cell line (CRL-1992; ATCC) pulsed with 10 mg/mL peptide at a ratio of 4:1 T2-to-T cells. IFNg spots were detected after 24 hours of stimulation and counted with an automated ELISpot Plate Reader (Autoimmun Diagnostika).
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