PBMCs from donors were seeded at a density of 1.5 × 105 cells/well in AIM-V medium (Gibco, Thermo Fisher Scientific) containing 2% human AB serum (Sigma-Aldrich) in 96-well U-bottom plates. Cells were stimulated with medium control (mock), AmiA, AliA, AliB, AliC or AliD at 1 μg/ml, in replicate wells per condition for 6 days at 37°C with 5% CO2 in the absence or presence of anti-MHC class II blocking antibodies consisting of a cocktail of anti-HLA-DR (B8.11-2; in-house produced), anti-HLA-DQ (SPV-L3; in-house produced) and anti-HLA-DP (B7/21; Leinco Technologies) monoclonal antibodies, as indicated. Tritium thymidine (18 kBq/well) was added to the wells on day six before overnight incubation at 37°C with 5% CO2. Cells were then harvested on a filter and incorporated label was determined as counts per minute (cpm) using a MicroBeta Counter (Perkin Elmer). Tritium thymidine based stimulation indices (SI) were calculated by dividing the mean cpm of the replicate stimulated wells, by the mean cpm of the quadruplicate medium control wells. SI’s > 1.7 were considered positive (van de Garde et al., 2019 (link)).
Microbeta counter
Manufactured by PerkinElmer
Sourced in United States
The MicroBeta counter is a high-performance liquid scintillation counter designed for accurate and efficient measurement of radioactive samples. It utilizes advanced detection technology to provide precise quantification of sample radioactivity.
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Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Optiphase supermix scintillation cocktail, by PerkinElmer (3 mentions)
3h thymidine, by PerkinElmer (2 mentions)
Triton x 100, by Merck Group (2 mentions)
3h rosiglitazone, by PerkinElmer (2 mentions)
Anti hla dr, by Leinco Technologies (1 mentions)
Anti hla dq, by Leinco Technologies (1 mentions)
Aim 5 medium, by Thermo Fisher Scientific (1 mentions)
Human ab serum, by Merck Group (1 mentions)
Optimem, by R&D Systems (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Zoledronic acid, by Merck Group (1 mentions)
Monensin, by Merck Group (1 mentions)
3h olmesartan, by American Radiolabeled Chemicals (1 mentions)
Catalase, by Merck Group (1 mentions)
L nna, by Merck Group (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
Na ve cd4 t cell isolation kit 2, by Miltenyi Biotec (1 mentions)
Filtermat a, by PerkinElmer (1 mentions)
Jes5 16e3, by BioLegend (1 mentions)
40 protocols using microbeta counter
1
Aliphatic Compounds Stimulate PBMC Proliferation
2
Anti-HIV Activity Screening of Drug Compounds
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TZM-bl cells were seeded on 96-well cell culture plates at a density of 1 × 104 cells/well and cultured overnight. Each drug was twofold serially diluted from 10,000 to 0.15 nM and treated to cells. After a 30 min incubation period, viral infection was performed with 500 TCID50 of HIV-1 AD8. Cells were cultured for 48 h after infection and luciferase activity measured using Beetle-Lysis Juice (PJK GmbH, Kleinblittersdorf, Germany) according to the manufacturer’s instructions. Briefly, the medium was removed, and cells washed three times with 200 μl PBS. Next, 100 μl Beetle-Lysis Juice containing luciferin and ATP were added to each well and incubated for 5 min with protection from sunlight. Subsequently, luciferase activity was measured using a micro beta counter (PerkinElmer, Waltham, MA) after transferring solutions to a white 96-well plate. Anti-HIV efficacy of drugs was determined based on reduced expression of luciferase relative to the virus-only treatment group.
3
Lymphocyte Proliferation Assay Across Species
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Swine (Yorkshire), bovine (Holstein and Black Angus), caprine (Boer Spanish cross), and ovine (Scottish Blackface) PBMCs (250,000 cells/well) were cultured in triplicate wells of 96 well round bottom plates for 24 hr. in a total volume of 100μL of cRPMI containing graded amounts of either mAb 2E4E4) or IgG1 isotype control (0.5, 1.0, 2.5, 5.0, or 10 μg/mL), PMA (1μg/mL)/Ionomycin (0.5μg/mL), or media alone. The cells were labeled for 12 hr. with 0.3 μCi of 3H-thymidine and incorporation of the isotope (in counts per minute) by the cells was determined using Microbeta Counter (PerkinElmer). Stimulation index (SI) was calculated from the CPM data for both 2E4E4 and IgG1 isotype control by dividing the treatment (2E4E4 or IgG1 isotype control) counts by the media control counts.
4
Mouse Splenocyte Proliferation Assay
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Spleens were harvested from mice and processed by lysing the RBCs with Gey’s lysis buffer. Single-cell suspensions were prepared, and cells were diluted to 4 × 106 cells/ml in complete culture medium (RPMI 1640 supplemented with 1% l -glutamine [100×], 1% penicillin-streptomycin, and 0.1% 2-mercaptoethanol with 10% heat-inactivated fetal bovine serum [FBS]). Samples were dispensed at 100 μl/well in triplicate and cultured with concanavalin A (ConA; 10 μg/ml), P. chabaudi AS, and P. yoelii YM pRBCs (5 × 106 pRBCs/ml), an equivalent concentration of nRBCs, or complete medium for 72 h at 37°C. Culture supernatants were removed and stored at −80°C for cytokine analysis and replaced with fresh complete medium. Cultures were pulsed with 1 μCi/well of [3H]thymidine (Perkin Elmer, Waltham, MA) after 54 h and left at 37°C for 18 h. Incorporation was stopped by storing the culture at −80°C, and the contents of the plates were harvested onto fiberglass mats. [3H]thymidine incorporation was measured on a PerkinElmer MicroBeta counter.
5
GTPγS Binding Assay for GPCR Activation
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GTPγS 35S binding experiments were performed by using cell-membrane homogenates as described. Membrane homogenates (10 μg) were equilibrated in a 200-μL volume of GTPγS 35S assay buffer (20 mmol/L HEPES, 10 mmol/L MgCl2, 100 mmol/L NaCl, 30 μg/mL saponin, and 0.1% bovine serum albumin, pH7.4) containing varying concentrations of CYM51010 and 10 μmol/L or 30 μmol/L guanosine diphosphate (MOR and DOR, respectively; 30 minutes, RT). After this time, 50 μL [35S] (0.3 nmol/L) was added, and incubation was continued for an additional 60 minutes (RT). Incubation was terminated by rapid filtration with a Packard plate harvester onto 96-well GF/C filter plates, followed by 3 washes with ice-cold Tris buffer (50 mmol/L Tris-HCl, 10 mmol/L MgCl2, 100 mmol/L NaCl, pH 7.6). After drying for 3 hours at 55°C, the GF/C filter plates were sealed with melt-on scintillator sheets. Bound [35S] was solubilized in 40 μL Microscint-20, and radioactivity was measured in a MicroBeta counter (Perkin-Elmer Life Sciences, Waltham, MA). Data were analyzed by using GraphPad Prism (San Diego) v8.0.1. Agonist concentration-response curves from GTPγS 35S experiments were fitted to the three-parameter logistic equation to derive estimates for agonist potencies (pEC50) and maximal agonist responses (Emax).
6
Allogeneic T cell proliferation assay
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0, 500, 5000 or 50 000 monocytes were co-cultured with 50 000 allogeneic naïve CD4+ T cells from healthy blood donors at indicated stimulator:responder ratios in OptiMEM (Gibco Life Technologies, Paisley, UK) supplemented with penicillin/streptomycin (Thermo Scientific, South Logan, Utah, USA), 10 ng/mL rhGM-CSF in all cultures and controls (added in order to improve cell survival as OptiMEM is nutrient-poor, R&D Systems, Minneapolis, MN, USA) and CD3/CD28 T cell activating dynabeads according to the manufacturer’s instructions (Gibco Life Technologies, AS, Oslo, Norway) for a total of 48h. 1 μCi [methyl-3H]thymidine was added for the last 18h and incorporation was measured in a Microbeta Counter (PerkinElmer, Boston, MA, USA). The background signal from monocytes was subtracted before calculating the relative proliferation of CD4+ T lymphocytes.
7
Evaluating Vγ9Vδ2 T Cell Cytotoxicity Against Ovarian Cancer
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For CD107a surface mobilization assay, primary EOC cells or cell lines were sensitized, or not, with 50 µM of zoledronic acid (Sigma Aldrich) overnight before incubation with allogeneic human Vγ9Vδ2 T lymphocytes (effector to target ratio 1:1). The coculture was performed in RPMI medium containing 5 µM monensin (Sigma) and Alexa Fluor 647-labeled anti-human CD107a mAb (#H4A3, Biolegend). After 4 h, human Vγ9Vδ2 T lymphocytes were collected and stained with a FITC-labelled anti-human TCR Vδ2 mAb (#IMMU389, Beckman Coulter) and analyzed by flow cytometry.
For cytolytic assays, EOC tumor cells, previously treated with zoledronate overnight, were incubated with5,r(75 µCi/106 cells) for 1 h. After washes, cells were cocultured with human Vγ9Vδ2 T lymphocytes (10:1) for 4 h.51,r release activity was measured in supernatants using a MicroBeta counter (Perkin Elmer). Percentage of target cell lysis = (experimental release – spontaneous release)/(maximum release – spontaneous release) x 100. Spontaneous release and maximum release were determined by adding medium or 1% Triton X−100, respectively, to1Cr-labelled target cells in the absence of human Vγ9Vδ2 T lymphocytes.
For cytolytic assays, EOC tumor cells, previously treated with zoledronate overnight, were incubated with5,r(75 µCi/106 cells) for 1 h. After washes, cells were cocultured with human Vγ9Vδ2 T lymphocytes (10:1) for 4 h.51,r release activity was measured in supernatants using a MicroBeta counter (Perkin Elmer). Percentage of target cell lysis = (experimental release – spontaneous release)/(maximum release – spontaneous release) x 100. Spontaneous release and maximum release were determined by adding medium or 1% Triton X−100, respectively, to1Cr-labelled target cells in the absence of human Vγ9Vδ2 T lymphocytes.
8
Monocyte and Fibroblast Proliferation Assay
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Primary human monocytes or primary mouse fibroblasts were cultured in different breast cancer conditioned media and allowed to proliferate for 24 h. [methyl-3H] thymidine (1 μCi) was added for 18 h, and incorporation was determined in a Microbeta Counter (Perkin & Elmer; MA, USA).
9
Cell Proliferation Assay Protocol
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Cell proliferation was determined after 5 days of in vitro stimulation by adding tritium thymidine (18 kBq/well) to the 96-well plates for overnight incubation at 37°C with 5% CO2 to be incorporated in the cellular DNA with every cell division. Cells were then harvested on a filter, and incorporated label was determined as counts per minute (cpm) using a MicroBeta counter (Perkin Elmer). Stimulation indices (SIs) were calculated by dividing the mean cpm of the quadruplicate stimulated wells by the mean cpm of the quadruplicate medium control wells. SIs of >1.7 were considered positive, and results are shown as mean SIs per group with standard deviations.
10
Chromium-release Cytotoxicity Assay for T-cells
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