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Prostaglandin extracts were prepared from supernatants and separated on silica gel/TLC plates before reading on a Bioscan plate reader.
After washing 4×, 100 µl of substrate (ABTS+H2O2) was added for 30 minutes at room temperature, before reading on a Versamax microplate reader at 405 nm.
Briefly, samples diluted at 1∶5 (50 µL final) were incubated with LAL reagent (50 µL) for 30 minutes at room temperature and a stop solution was added before reading on a spectrophotometer at 405 nm.
Finally, cells were fixed with 1% paraformaldehyde before reading on a flow cytometer.
Plates were revealed with tetramethylbenzidine, and the reaction was stopped with 1 M sulfuric acid before reading on a spectrophotometer (Multiskan Ex, Thermo Scientific) at 450 nm.
After washing, 20 µl/well of ELISA Pico Chemiluminescent Substrate Thermo Fisher Piercee) was added and incubated for 5 min before reading on a plate reader.
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The wells were then washed, incubated with recombinant αv β3 for 1.5 h, then washed and incubated with anti- αv β3 antibody for 1.5 h, then washed and incubated with anti-mouse-HRP-conjugated secondary antibody for a further hour and finally washed and incubated with OPD for 15 min before reading on an ELISA plate reader (450 nm).
If not this or anything else we said before, read on to the next step.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com