Cells were fixed in 4% paraformaldehyde for 10 minutes and then washed in phosphate buffered saline with 0.1% Triton X-100 (PBS-T). Primary antibodies (Table S4 ) were diluted in immunobuffer (PBS containing 1% goat or donkey serum, 0.2% Triton X-100 and 0.04% thimerosal) and incubated with cells overnight at 4 °C. Cells were washed in PBS-T and then incubated with the appropriate fluorescent secondary antibodies (Table S4 ) for 4 hours at room temperature. Cells were washed again, and cell nuclei were counterstained with Hoechst 33258 (Sigma-Aldrich, MO, USA) for 20 min. Images were acquired at 10X magnification on an ImageXpress Micro XLS automated fluorescence microscope (Molecular Devices, CA, USA). Integrated intensity measures (VCAM-1) and percentage of total cells that stained positively (MCP-1) were quantified using the MultiWavelength Cell Scoring module within MetaXpress software (Molecular Devices, CA, USA).
Multi wavelength cell scoring module
Manufactured by Molecular Devices
Sourced in United States
The Multi-Wavelength Cell Scoring module is a laboratory instrument designed to measure and analyze multiple characteristics of cells. It utilizes various wavelengths of light to assess cellular properties such as size, morphology, and fluorescence intensity. This module provides quantitative data to support research and analysis in diverse fields, including cell biology, drug discovery, and diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
Microtiter plates, by PerkinElmer (2 mentions)
Metaexpress image analysis software, by Molecular Devices (2 mentions)
Metaxpress software, by Molecular Devices (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Alexa 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Alexa 647 secondary antibodies, by Thermo Fisher Scientific (1 mentions)
3 protocols using multi wavelength cell scoring module
1
Quantifying Immunofluorescence Markers
2
High-Throughput Melanoma Phenotypic Screening
3
High-throughput Apoptosis Screening Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Approximately 250 cells per well for each cell line were seeded in each well of 1,536-well microtiter plates (PerkinElmer) and incubated overnight. Combinatorial drug library was prepared as 300X stocks in 384 well plate and 20nL pin-transferred to 1,536-well plates. Plates were incubated for 72 hours and fixed in 4% formaldehyde, washed in PBS containing 0.1% Triton, and incubated overnight with antibodies (1:500) to cleaved PARP (above). The plates were then washed twice, incubated with Alexa 647 secondary antibodies (1:1000, Life Technologies) and DAPI 2–16 hours, and washed with PBST. Plates were then imaged using the Cellworx high-throughput microscope (Applied Precision Inc.) and counting nuclei and cells with cPARP staining with the Multi-Wavelength Cell Scoring module of the MetaExpress image analysis software (Molecular Devices). Z’ values were between 0.8–0.9 for most cell lines.
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