CD8+ cells were sorted as above from primed WT mice, healthy donor leukopaks or hCLL PBMCs. Mouse T-cells were cultured at 0.75x106/mL for three days with soluble 10μg/mL anti-CD3 and/or Ficoll-Paque purified Eμ-TCL1 splenocytes. Human T-cells were cultured at 1x106/mL for five days with plate-bound 10μg/mL anti-CD3 and soluble 1μg/mL anti-CD28. Cells were then pulsed with 1μCi 3H-thymidine (Perkin Elmer, Waltham, MA) or 40μM BrdU for four hours at 37°C. Counts from 3H-thymidine incorporation were measured with a TopCount NXT Microplate Scintillation & Luminescence Counter (Perkin Elmer) and BrdU incorporation was measured on an LSR II or Symphony PRO flow cytometer (BD Biosciences).
Topcount nxt microplate scintillation luminescence counter
Manufactured by PerkinElmer
The TopCount NXT Microplate Scintillation & Luminescence Counter is a versatile instrument designed for high-throughput detection of radioactive and luminescent samples in microplate format. It features a modular design, multi-detection capabilities, and automated sample handling to support a wide range of applications in life science research and drug discovery.
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Lab products found in correlation
5 protocols using topcount nxt microplate scintillation luminescence counter
1
CD8+ T-cell Proliferation Assay
2
CD8+ T-cell Proliferation Assay
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CD8+ cells were sorted as above from primed WT mice, healthy donor leukopaks or hCLL PBMCs. Mouse T-cells were cultured at 0.75x106/mL for three days with soluble 10μg/mL anti-CD3 and/or Ficoll-Paque purified Eμ-TCL1 splenocytes. Human T-cells were cultured at 1x106/mL for five days with plate-bound 10μg/mL anti-CD3 and soluble 1μg/mL anti-CD28. Cells were then pulsed with 1μCi 3H-thymidine (Perkin Elmer, Waltham, MA) or 40μM BrdU for four hours at 37°C. Counts from 3H-thymidine incorporation were measured with a TopCount NXT Microplate Scintillation & Luminescence Counter (Perkin Elmer) and BrdU incorporation was measured on an LSR II or Symphony PRO flow cytometer (BD Biosciences).
3
TGF-β1-Induced Chondrogenesis Assay
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Chondrogenic differentiation was assessed in high-density (0.5-1 × 105 cells per well) cultures in 96-well plates with 10 ng/ml TGF-β1 for 48 hours as described previously [19 (link)]. Glycosaminoglycan synthesis was measured in quadruplicate wells by 35SO4 incorporation using a TopCount NXT Microplate Scintillation & Luminescence counter (Perkin Elmer Life and Analytical Sciences, Downers Grove, IL). Values for GAG synthesis were normalised to DNA content per well.
4
CTL-Mediated Cytotoxicity Assay
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Target cells were incubated with respective QW9 and QW9 variant peptides at a concentration of 10 μg/mL and incubated with 51Cr at a concentration of 250μCi/mL for one hour in a 37 °C and 5% CO2 incubator. 51Cr-labeled cells were washed 3 times in RPMI media with 10% FBS and resuspended at a concentration of 1 million cells/mL. Target cells were plated in a flat-bottom 96-well plate. To ensure an equal number of QW9-specific cells, tetramer-positive cells were sorted to be used in the killing assay and were added at the appropriate effector-target ratios. Spontaneous and maximum releases were determined by incubating the labeled target cells with medium alone or 5% Triton X-100 (Cat. A16046.AP, ThermoFisher), respectively. The supernatant was collected after 6 hours of incubation at 37 °C in 5% CO2. We used a Perkin Elmer TopCount NXT Microplate Scintillation & Luminescence Counter to measure the radioactivity present in the supernatant. Quantification of specific killing was calculated as specific killing =100 x (sample release – spontaneous release) / (maximum release - spontaneous release).
5
Receptor Binding Assay in Rat Synaptic Membranes
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Receptor binding assay in rat synaptic membranes.
Rat (Sprague-Dawley) cortical synaptic membranes were prepared as previously described. 38 The membranes were stored at -20 °C until the day of assay where they were washed four times with 50 mM Tris-HCl buffer (pH 7.4). The binding protocol was performed in 96-well microplates, as previously described. 29 In brief, compounds were incubated with [ 3 H]muscimol and membranes at 0-4 °C, filtered through GF/C filter plates (PerkinElmer) using a 96-well harvester (Packard), rapidly washed three times with ice-cold binding buffer, and filter plates dried. CPM values were determined using liquid scintillation counting in a TopCount NXT Microplate Scintillation & Luminescence Counter (PerkinElmer). CPM values were fitted by non-linear regression using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA), and IC50 values determined and converted to Ki values using the Cheng-Prusoff equation. The experiments were performed in triplicate at least three times for each compound. Non-specific binding was determined using 1.0 mM GABA. 39
Rat (Sprague-Dawley) cortical synaptic membranes were prepared as previously described. 38 The membranes were stored at -20 °C until the day of assay where they were washed four times with 50 mM Tris-HCl buffer (pH 7.4). The binding protocol was performed in 96-well microplates, as previously described. 29 In brief, compounds were incubated with [ 3 H]muscimol and membranes at 0-4 °C, filtered through GF/C filter plates (PerkinElmer) using a 96-well harvester (Packard), rapidly washed three times with ice-cold binding buffer, and filter plates dried. CPM values were determined using liquid scintillation counting in a TopCount NXT Microplate Scintillation & Luminescence Counter (PerkinElmer). CPM values were fitted by non-linear regression using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA), and IC50 values determined and converted to Ki values using the Cheng-Prusoff equation. The experiments were performed in triplicate at least three times for each compound. Non-specific binding was determined using 1.0 mM GABA. 39
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