A mixture of 5 μg protein membrane and 1 mg SPA bead was pre-coupled in a shaker (Vibrax VXR, IKA) in a volume of 50 μL of assay buffer at room temperature for 30 min. Then, together with the radioligand and competing ligands, the membrane-bead mixture was dispatched in an Isoplate-96 Microplate (Perkin Elmer, Groningen, the Netherlands), in a final reaction volume of 100 μL. The plate was incubated for 1 h inside the counting chamber of a 2450 MicroBeta2 Plate Counter (Perkin Elmer, Groningen, the Netherlands) at the ambient temperature of 28 °C. The binding values were recorded in corrected counts per minute (CCPM).
2450 microbeta2 plate counter
Manufactured by PerkinElmer
Sourced in United States
The 2450 MicroBeta2 Plate Counter is a microplate reader designed for high-throughput scintillation and luminescence detection. It is capable of measuring radioactive and luminescent samples in microplates. The device provides precise and reliable measurements for a variety of applications in life science research and drug discovery.
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Lab products found in correlation
6 protocols using 2450 microbeta2 plate counter
1
Membrane-Bead Binding Assay Protocol
2
Radioligand Displacement Assay Protocol
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Radioligand displacement
experiments were performed as in previously published methods.7 (link) Membrane aliquots containing 15 μg of protein
were incubated in a total volume of 100 μL assay buffer (50
mM Tris–HCl, 5 mM MgCl2, supplemented with 0.01%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and 1 mM
ethylenediaminetetraacetic acid (EDTA), pH 7.4) at 25 °C for
120 min. Displacement experiments were performed using six concentrations
of competing antagonist in the presence of ∼10 nM [3H]PSB-11. Nonspecific binding was determined in the presence of 100
μm NECA and represented less than 10% of total binding. Incubation
was terminated by rapid filtration performed on 96-well GF/B filter
plates (PerkinElmer, Groningen, the Netherlands) in a PerkinElmer
Filtermate-harvester (PerkinElmer, Groningen, the Netherlands). After
the filter plate was dried at 55 °C for 30 min, the filter-bound
radioactivity was determined by scintillation spectrometry using a
2450 MicroBeta2 Plate Counter (PerkinElmer, Boston, MA).
experiments were performed as in previously published methods.7 (link) Membrane aliquots containing 15 μg of protein
were incubated in a total volume of 100 μL assay buffer (50
mM Tris–HCl, 5 mM MgCl2, supplemented with 0.01%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and 1 mM
ethylenediaminetetraacetic acid (EDTA), pH 7.4) at 25 °C for
120 min. Displacement experiments were performed using six concentrations
of competing antagonist in the presence of ∼10 nM [3H]PSB-11. Nonspecific binding was determined in the presence of 100
μm NECA and represented less than 10% of total binding. Incubation
was terminated by rapid filtration performed on 96-well GF/B filter
plates (PerkinElmer, Groningen, the Netherlands) in a PerkinElmer
Filtermate-harvester (PerkinElmer, Groningen, the Netherlands). After
the filter plate was dried at 55 °C for 30 min, the filter-bound
radioactivity was determined by scintillation spectrometry using a
2450 MicroBeta2 Plate Counter (PerkinElmer, Boston, MA).
3
Quantifying T Cell Cytotoxicity and IFNγ Secretion
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For IFNγ secretion, 5.000 T cells were incubated with target cells in varying E:T ratios in 384-well plates (Greiner Bio-One, Austria) overnight in 60ul of TCM. 12.5ul of supernatants were harvested and serially diluted at 1:5 and 1:25. IFNγ was measured using IFNγ ELISA (Sanquin, The Netherlands). Values of IFNγ concentrations were back calculated accounting for respective dilution factors. If values were outside of the linear range of the standard in the 1:5 dilution, values generated with the 1:25 dilution were used for figures.
To assess cytotoxicity as measured by chromium release, target cells were labeled with 100 μCi Na251CrO4 for 1 h at 37°C and washed 3 times. Labeled target cells were then incubated in triplicate with T cells in varying E:T ratios in TCM for 6 h at 37°C. 25ul of respective supernatants were then harvested, transferred to 96-well LumaPlates (PerkinElmer, US), and allowed to dry overnight. Chromium release was measured using a 2450 Microbeta2 plate counter (PerkinElmer, US). Toxicity was calculated using the following formula
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% ( k i l l i n g ) = t e s t r e l e a s e − s p o n t a n e o u s r e l e a s e m a x i m u m r e l e a s e − s p o n t a n e o u s r e l e a s e ∗ 100.
To assess cytotoxicity as measured by chromium release, target cells were labeled with 100 μCi Na251CrO4 for 1 h at 37°C and washed 3 times. Labeled target cells were then incubated in triplicate with T cells in varying E:T ratios in TCM for 6 h at 37°C. 25ul of respective supernatants were then harvested, transferred to 96-well LumaPlates (PerkinElmer, US), and allowed to dry overnight. Chromium release was measured using a 2450 Microbeta2 plate counter (PerkinElmer, US). Toxicity was calculated using the following formula
4
Proliferative Response to Autoantigen GAD65
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Proliferative response was analyzed in PBMCs cultured in triplicates in AIM-V medium with β-mercaptoethanol at 37°C in 5% CO2, in the presence of 5 μg/mL rhGAD65 (Diamyd Medical, Stockholm), CD3/CD28 beads (∼1 bead: 2 cells; Gibco, Life Technologies AS, Oslo, Norway) or buffer alone (Diamyd Medical, Stockholm, Sweden) (18 (link)). After 3 days, cells were pulsed for 18h with 0.2 µCi of [3H] thymidine/well (PerkinElmer), and thereafter 3H incorporation was recorded using a 2450 MicroBeta 2 Plate Counter (PerkinElmer, Waltham, MA, USA). Proliferation was expressed as stimulation index (SI), calculated as the mean of triplicates in the presence of stimulus, divided by the mean of triplicates in buffer alone.
5
Evaluating EZH1/2 Inhibitors for HAM Treatment
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PBMCs from patients with HAM (n = 8) were seeded at 1 × 105 cells/well in 96-well round bottom plates. They were cultured for 7 days in the presence or absence of OR-S1, valemetostat, GSK126, tazemetostat, or prednisolone. During the last 16 h, 1 μCi of 3H-thymidine was added to each well, and then the cells were harvested and counted using a 2450 MicroBeta2 Plate Counter (Perkin Elmer, Boston, MA). The assay was performed in triplicate. To verify the effect of the EZH1/2 inhibitors, PBMCs from another eight patients with HAM were also used. Using the average counts of 3H-thymidine incorporation in the DMSO-treated group as 100%, the average of the relative values in each drug-treated group was calculated and defined as the 3H-thymidine incorporation rate (%). The IC50 of each drug was calculated by substituting the data obtained from this experiment into the following equation: IC50 = 10ˆ[LOG(A/B)∗(50-C)/(D-C) + LOG(B)], where A is the higher concentration considering the two values that sandwich 50% of 3H-thymidine incorporation rate, B is the lower concentration considering the same two values, C is the 3H-thymidine incorporation rate (%) determined for B, and D is the 3H-thymidine incorporation rate (%) determined for A.
6
Displacement Assay for A3 Adenosine Receptor
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