Cells were seeded into 384-well tissue culture plates (PerkinElmer, Waltham, MA, USA, #6007689) at a density of 1000 cells/well in 20 uL of media and were allowed to attach overnight. The next day, drugs were pre-diluted at 4x final concentration in culture medium then added to the 384-well plates so that the final volume was 40 μL/well. For the single compound dose response experiments, the compound was pre-diluted at 2x the final concentration and 20 uL was added to each well. A PBS or growth medium barrier was added to the outer wells of the plate to limit evaporation. Cells were cultured under these conditions for 72 h. To assess viability, 10 μL of CellTiter-Glo (Promega, Madison, WI, USA, #G7573) was added to each well. Plates were incubated for 5 min at room temperature then briefly centrifuged (4000 rpm, 60 seconds) before being read on a Bio-Tek Synergy Neo plate reader. Viability signal is plotted versus log(Vemurafenib concentration) for each treatment condition. The Area Under the Curve (AUC) was calculated for each curve using GraphPad Prism for the range log concentration from −9 to −5.
384 well tissue culture plates
Manufactured by PerkinElmer
Sourced in United States
The 384-well tissue culture plates are a versatile laboratory equipment designed for cell culture and high-throughput screening applications. They provide a standardized format with a high density of individual wells, allowing for the efficient use of limited sample volumes and reagents. The plates are made of materials suitable for cell culture and are compatible with various automated liquid handling systems and plate readers.
Automatically generated - may contain errors
5 protocols using 384 well tissue culture plates
1
Cell Viability Assay with Vemurafenib
2
Cell Viability Assay with Vemurafenib
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Cells were seeded into white 384-well tissue culture plates (PerkinElmer, Waltham, USA, #6007689) at a density of 1000 cells/well in 20 µL of growth medium. The next day, compounds were pre-diluted in growth medium and then added to the 384-well plates so that the final volume of each well was 40 µL. The outer wells of the plate received no cells, and a PBS or growth medium barrier was added to those wells to limit evaporation from wells containing cells. Cells were cultured under these conditions for 72 h. To assess viability, 8 µL of CellTiter-Glo (Promega, Madison, USA, #G7573) was added to each well. Plates were incubated on an orbital shaker for 5 min at room temperature, then briefly centrifuged (4000 rpm, 60 s) before being read on a Bio-Tek Synergy Neo plate reader with the #11 and #41 Ex/Em filter cubes. Viability signal was plotted versus the log of the vemurafenib concentration for each treatment condition.
3
Screening siRNA Library for KRAS Inhibition
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Custom-made siRNA library assay plates were reconstituted according to manufacturer’s instructions (Dharmacon). NCI-H2030 cells were reverse transfected onto 384-well tissue culture plates (Perkin Elmer) using RNAi max (Invitrogen Thermofisher Scientific) at a final siRNA concentration of 20 nM. Cells were washed after 6 h and fresh media added. After 72 h, cells were treated with varying concentrations of AZD4785, left for 48 h before being processed for RT-qPCR as described previously. KRAS gene expression was normalized to housekeeping gene 18S rRNA and shown relative to PBS control. Data were plotted using GraphPad Prism Software; for determining siRNA hits based on IC50, dose–response curves were plotted, and data were analysed using non-linear regression and curves were fit using log (inhibitor) versus response, variable slope (four parameters) equation using GraphPad Prism, curves were constrained between 0 and 1. For determining siRNA hits based on maximum inhibition, dose–response curves were plotted, and data were analysed using non-linear regression. For siRNA validation experiments, siRNA On-Target SMARTpool and single oligonucleotides were obtained from Dharmacon and reconstituted according to manufacturer’s instructions.
4
Cell Viability Assay of Drug Treatments
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Cells were seeded into 384-well tissue culture plates (PerkinElmer (Waltham, MA, USA), #6007689) at a density of 1000 cells overnight. On the next day, increasing concentrations of drugs were added using a pin tool (~150 nL) and incubated for 72 h. To measure cell viability, CellTiter-Glo (Promega (Madison, WI, USA), G7573) was added, and plates were read on a Bio-Tek Synergy Neo-plate reader according to the manufacturer’s protocol. Cell viability readings were normalized to DMSO-treated cells. Data were plotted by GraphPad Prism (San Diego, CA, USA) using drug dose–response curves, and IC50 was calculated.
5
Cell Viability Assay with Vemurafenib
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Cells were seeded into 384-well tissue culture plates (PerkinElmer, Waltham, MA, USA, #6007689) at a density of 1000 cells/well in 20 uL of media and were allowed to attach overnight. The next day, drugs were pre-diluted at 4x final concentration in culture medium then added to the 384-well plates so that the final volume was 40 μL/well. For the single compound dose response experiments, the compound was pre-diluted at 2x the final concentration and 20 uL was added to each well. A PBS or growth medium barrier was added to the outer wells of the plate to limit evaporation. Cells were cultured under these conditions for 72 h. To assess viability, 10 μL of CellTiter-Glo (Promega, Madison, WI, USA, #G7573) was added to each well. Plates were incubated for 5 min at room temperature then briefly centrifuged (4000 rpm, 60 seconds) before being read on a Bio-Tek Synergy Neo plate reader. Viability signal is plotted versus log(Vemurafenib concentration) for each treatment condition. The Area Under the Curve (AUC) was calculated for each curve using GraphPad Prism for the range log concentration from −9 to −5.
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