C166 and MS1 endothelial cells (2,000 cells/well) were incubated in the presence of rTGF-β1, for the duration indicated in the figure legends. One µCurie of [3H]-thymidine (3H-T) was added during the last 24 h of culture. On the day of revelation, cells were incubated with trypsin and aspirated on a 96-filter plate (Unifilter GF/C) using a harvester (Packard Filtermate 196). The plate was washed several times with water. Radioactivity was measured by a scintillation counter (Packard Microplate Scintillation Counter), which calculates the counts per minute (cpm).
Filtermate 196
Manufactured by Hewlett-Packard
Sourced in United States
The Filtermate 196 is a laboratory filtration device designed for the efficient separation of solid particles from liquids. It features a durable construction and accommodates various filter media to suit diverse filtration requirements. The core function of the Filtermate 196 is to facilitate reliable and consistent liquid filtration processes in a laboratory setting.
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2 protocols using filtermate 196
1
Proliferation Assay of Endothelial Cells
2
Cell Proliferation Quantification by Radiolabeled Thymidine
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Cell proliferation was assessed by measuring 3 H-labelled thymidine incorporation as a surrogate of DNA replication. LARA cells were seeded in 96-well plates (Cellstar; Greiner Bio-One) in medium containing 10% DCC-FCS. The next evening, media were replaced by different percentages of human serum supplemented with 3 H-thymidine (PerkinElmer, Waltham, MA, U.S.A.). Sixteen hours later, media were removed and cells were rinsed, followed by trypsinization and transfer to a new multiwell plate using a cell harvester (Filtermate 196, Packard, Meriden, CT, U.S.A.). Fifty microlitres of liquid scintillation fluid (Microscint-O, Packard) was added to each well and disintegrations were counted using a microplate scintillation counter (Packard Topcount) as described previously (Helsen et al., 2012) . Measurements were corrected for background in wells without cells (<1%) as well as values of input media used for stimulation, and expressed as percentage of the 5% serum condition.
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