Cryopreserved hPD-PBMCs were rapidly thawed, washed twice in PBMC media, and resuspended at 1.5 × 106 PBMC per mL of PBMC media as above. In a 6-well plate, 4.5 × 106 PBMCs in 2.5 mL were placed per well and treated as control (no addition), mitogen stimulated (15 µg/mL PHA-P with the addition of 7.5 µL of 5 mg/mL PHA-P) or apoptosis-induced (1 µM staurosporine with the addition of 2.5 µL of 1 mM staurosporine in DMSO; Sigma Aldrich Cat # S6942). At 24 and 48 h, PBMCs were resuspended and analyzed for viability, phosphatidylserine externalization, and caspase 3/7 activity.
S6942
Manufactured by Merck Group
Sourced in Germany
S6942 is a laboratory equipment product offered by Merck Group. It serves as a device for scientific research and analysis, though its core function is not explicitly stated in the instructions provided.
Automatically generated - may contain errors
Lab products found in correlation
Spark 10m, by Tecan (1 mentions)
Alamar blue, by Thermo Fisher Scientific (1 mentions)
Z vad fmk, by BD (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
Staurosporine, by Merck Group (1 mentions)
G3580, by Promega (1 mentions)
Celltiter 96 aqueous non radioactive cell proliferation assay, by Promega (1 mentions)
Multiskan ascent spectrophotometer, by Thermo Fisher Scientific (1 mentions)
4 protocols using s6942
1
Apoptosis Induction in Cryopreserved PBMCs
2
Cell Viability Assay with Apoptosis Modulators
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Cells were seeded at a concentration of 3 × 103 cells per well in 96 well plates with or without the indicated concentrations of proteins in triplicate. When indicated, zVAD-fmk, a general caspase inhibitor (BD Biosciences) and staurosporine, an initiator of apoptosis (S6942, 1 mM stock solution, Sigma-Aldrich) were used at a 50 and 1 μM final concentration. After incubation at 27 °C for the indicated time, 0.1 volume of Alamar Blue (Invitrogen) was added and the cells were incubated at 27 °C for an additional 3 h. Fluorescence (excitation, 530–560 nm; emission, 590 nm) was measured using a microplate reader (TECAN spark 10 M). Results are presented as a percentage of viable cells relative to experimental control (set as 100%).
3
Cell Viability and Migration Assay
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Cells were seeded into a 96-well plate with MDA-MB-231 cells 2.000/well and HeLa cells 1.600/well. Protein mixtures were added (100 µL/well) as indicated and the cells were incubated at 37 °C, accordingly. As control, either DMSO (20%) or staurosporine (1 µM, S6942, Sigma-Aldrich Chemie, Taufkirchen, Germany) was added to the cells. To determine cell viability, the Cell Titer 96 AQueous One Solution cell proliferation assay [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay] from Promega (#G3580, Promega, Madison, WI, USA) was used. Ten microliters of MTS substrate was added and the cells further incubated for 90 min at 37 °C. The absorbance was measured at 495 nm. Signal intensity was normalized to untreated cells for statistical analysis. In case of the migration assay, a total volume of 25 µL of MTS substrate was added. After 90 min of incubation at 37 °C, 100 µL of each well were transferred into a 96-well plate and the absorbance measured as described above and earlier [32 (link)].
4
Quantifying Primary Neuron Viability
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Cell viability was measured by the ability to metabolize 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS, CellTiter 96® AQueous non-radioactive cell proliferation assay, G5421, Promega) in the presence of the electron coupler phenazine methosulfate (PMS) to a media soluble formazan product, as described previously [9 (link)]. Combined MTS (400 μg/ml) and PMS (44 μg/ml) solution was incubated on primary cultured neurons for 4 h (37 °C). Culture medium absorbance at 492 nm was then determined using a Multiskan Ascent spectrophotometer (Thermo Scientific). All sample absorbance readings were then blank normalized using a negative control reaction containing only culture medium and the MTS/PMS reagent. Absorbance readings of all treatment groups were then normalized back to genotype-specific vehicle controls and expressed as a percentage of vehicle control cell viability. Experiments were performed in technical triplicate and staurosporine (1 μM, S6942, Sigma) was used to induce cellular apoptosis in primary neuronal cultures for positive control means.
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