A proliferation assay was performed by culturing purified CD3 cells (3 × 106) from IRBP1–20‐immunized B6 mice at 37°C for 48 h in 96‐well microtiter plates in complete medium containing graded doses of the immunizing peptide in the presence of BMDCs (1.5 × 105/well) under Th1 polarizing conditions (culture medium supplemented with 10 ng/mL of IL‐12) or Th17 polarizing conditions (culture medium supplemented with 10 ng/mL of IL‐23) in a total volume of 200 µL. [3H] thymidine incorporation during the last 8 h was assessed using a microplate scintillation counter (Packard). The proliferative response was expressed as the mean cpm ± standard deviation (SD) of triplicate determinations.
Microplate scintillation counter
Manufactured by Hewlett-Packard
Sourced in United States
The Microplate Scintillation Counter is a laboratory instrument designed to measure the radioactivity levels in samples contained within microplates. It is capable of detecting and quantifying low-level radioactive signals, making it a useful tool for various applications in scientific research and analysis.
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8 protocols using microplate scintillation counter
1
CD3 Cell Proliferation Assay
2
Antigen-Specific T Cell Proliferation Assay
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As modified from our previously published protocol,[10 (link)] CD8 or CD4 enriched T cells from MOG35–55-immunized wildtype B6 mice were prepared and seeded at 4 × 105 cells/well in 96-well plates. The cells were then cultured at 37°C for 48 h in 200 μL medium with or without MOG35–55 in the presence of mitomycin C-treated syngeneic spleen antigen presenting cells (1 × 105). [3H] thymidine incorporation during the last 8 h was assessed by a microplate scintillation counter (Packard; PerkinElmer, Meriden, CT, USA). The proliferative response is expressed as the mean counts per minute ± standard deviation (SD) of triplicate determinations.
3
Allogeneic Mixed Lymphocyte Culture Assay
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MLC was carried out by incubating 1.25 × 106 responder spleen cells/ml with an equal number of irradiated (30 Gy from a 137 Cs source) adherent spleen cells. In some cases, responders were CD4+, CD8+, and CD5+ cells purified from spleens and seeded at a density of 0.75 × 106 cells/ml. Adherent cells were obtained by coating 1 × 106 splenocytes in a 96‐well flat bottom microtiterplate for 1.5 h and removing non‐adherent cells by washing the microplate twice with PBS (37°C). DC subpopulations were cultured at 104 cells per well. Proliferation was measured after two days by incubation with 3H‐thymidine at 1 µCi (0.037 MBq)/well for a further 18 h. 3H‐thymidine incorporation was measured using a scintillation counter (Packard Microplate Scintillation Counter). MHC II activity was blocked in vitro using anti‐MHC II LEAF anti‐mouse I‐A/I‐E clone M5/114.15.2 (Biolegend, San Diego, CA) at 5 µg/ml.
4
PBMC Stimulation and Treg Expansion
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PBMC at ≤106 cells/mL in growth medium were incubated in quadruplicate wells of 96-well microplates (Corning) at 100μL/well in the presence of 100 μL of inactivated CMV-infected cell lysate, inactivated varicella zoster virus (VZV)-infected cell lysate, C. albicans antigen (Greer), HIV inactivated virion [26 (link)], the appropriate mock-infected cell control antigens and phytohemagglutinin (PHA; Sigma) positive control. After 6 days of antigenic or mitogenic stimulation at 37°C, in a 5% CO2 and 95% H2O atmosphere, cells were pulsed with 1 μCi of 3H-thymidine and harvested 6 h later onto Unifilter plates (Perkin Elmer). Radioactivity gathered on the filters was counted in scintillation fluid (Perkin Elmer) with a microplate scintillation counter (Packard). Results were expressed in median counts per minutes (cpm) of the quadruplicate wells. Inhibition was measured on samples that showed ≥3-fold increase of median cpm in antigen- compared with mock-stimulated wells.
Treg expansion with rhIL2 (30ng/mL) was carried out in 96-well microtiter plates as described above for up to 15 days, during which rhIL2 and growth medium were replenished twice weekly. At the end of the expansion, viable cells were counted with a Guava 8HT instrument.
Treg expansion with rhIL2 (30ng/mL) was carried out in 96-well microtiter plates as described above for up to 15 days, during which rhIL2 and growth medium were replenished twice weekly. At the end of the expansion, viable cells were counted with a Guava 8HT instrument.
5
IRBP-Specific T Cell Proliferation
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Purified αβ T cells (3×105 cells/well) from IRBP1-20-immunized B6 mice in a total volume of 200 µl were cultured at 37°C for 48 h in 96-well microtiter plates in complete medium with or without 10 µg/ml of immunizing peptide in the presence of irradiated syngeneic spleen APCs (2×105), and [3H] thymidine incorporation during the last 8 h was assessed using a microplate scintillation counter (Packard). The proliferative response was expressed as the mean cpm ± standard deviation (SD) of triplicate determinations.
6
Proliferation Assay of Endothelial Cells
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C166 and MS1 endothelial cells (2,000 cells/well) were incubated in the presence of rTGF-β1, for the duration indicated in the figure legends. One µCurie of [3H]-thymidine (3H-T) was added during the last 24 h of culture. On the day of revelation, cells were incubated with trypsin and aspirated on a 96-filter plate (Unifilter GF/C) using a harvester (Packard Filtermate 196). The plate was washed several times with water. Radioactivity was measured by a scintillation counter (Packard Microplate Scintillation Counter), which calculates the counts per minute (cpm).
7
Nylon Wool-Enriched T Cell Proliferation
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Nylon wool-enriched T cells, prepared from aEAU or tEAU rats were seeded at 4 × 10 5 cells/well in 96-well plates and cultured at 37°C for 60 h in a total volume of 200 μl culture medium with or without 10 g/ml of R16 in the presence of irradiated syngeneic spleen antigen presenting cells (APCs) (1×10 5 ). In some experiments, additional exosomes at different concentrations were given. ( 3 H)thymidine incorporation during the last 16 h, was assessed using a microplate scintillation counter (Packard). The proliferative response was expressed as the mean cpm ± standard deviation (SD) of triplicate determinations. We also used BrdU assay kits (Roche) for T cell proliferative response which was expressed as the mean OD ± SD for triplicate This article is protected by copyright. All rights reserved.
samples or the proliferative stimulus index calculated as the ratio of the mean OD measured in the presence of the R16 to that in the absence of R16 (21).
samples or the proliferative stimulus index calculated as the ratio of the mean OD measured in the presence of the R16 to that in the absence of R16 (21).
8
Cr Release Assay for BALB/c Mice Immunized with MFP
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Cr release assay BALB/c mice immunised (x 3) with MFP (5 μg) were culled and their spleen cells collected and treated with 0.83% NH4Cl. Two-fold serial dilutions of effector spleen cells from the immunised mice were plated into a 96 well plate beginning at a concentration of 1 x 10 6 cells per well in duplicate. 1 x 10 4 51 Cr labelled DA3-MUC1 cells cultured with IFN-γ (20 ng/ml) for 72 h, DA3-MUC1, P815-MUC1 or P815 target cells were added to the effectors. The spontaneous release of 51 Cr from the labelled cells was determined by incubating target cells in RPMIM media and the maximum release was determined by incubation with 10% SDS (BDH Chemicals, Dorset, England). Cultures were incubated for 4 h before transferring 100 μl of supernatant to 96 well flat Optiplates (Disposable Products, Australia) containing 100 μl of Microscint 40 (Packard, USA) for analysis on the microplate scintillation counter (Packard USA). The specific percentage lysis of target cells was determined by; [(experimental-spontaneous) cpm / (maximum-spontaneous) cpm] x 100%.
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