Flow cytometry Anti-bodies Human - CD31/PECAM-1

Flow cytometry is an immunophenotyping technique whereby sing cell suspensions are stained for either cell surface markers or intracellular proteins by fluorescently-labelled antibodies and analyzed with a flow cytometer, where fluorescently-labelled molecules are excited by the laser to emit light at varying wavelengths, which is then detected by the instrument. There are several key criteria which are required to be kept in mind while designing a flow experiment- 1. Antibody titration (optimal dilution of antibodies should be calculated in order to avoid over- or under- saturated signals for proper detection of surface and intracellular markers), 2. Precision (3 or more replicates of the sample should be used per experiment), 3. Specificity (proper isotype controls should be included in the experiment), 4. Day-to-day variability (experiments should be repeated 3 or more times to ensure consistency and avoid variability due to flow cytometer settings), 5. Antibody interaction (Fluorescence minus one or FMO should be used, which is the comparison of signals from panel minus one antibody vs. the full panel), and 6. Antibody stability (fluorescently-labelled antibodies should be stored at 4C).

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Found 3 matching solutions for this experiment

CD31-FITC, 5.6E, 2 mL, ASR

Beckman Coulter

Upstream tips	Protocol tips	Downstream tips
Endothelial cells were stimulated with P. aeruginosa PAO1 strain and QSSMs (20 µM) for 20 hours and the expression of cell adhesion and inflammatory markers was assessed by flow cytometry (Gallios, Beckman-Coulter) using 1 × 105 endothelial cells stained	Endothelial cells were detached using trypsin (Sigma-Aldrich, USA) and washed in PBS. Cells were then incubated with the primary antibodies at room temperature in the dark for 30 min. Further, the cells were washed twice and centrifuged at 400g, 10 min, in PBS suplemented with 1% BSA.	Flow cytometry data were analyzed using the Gallios software (Beckman-Coulter).

Upstream tips
Endothelial cells were stimulated with P. aeruginosa PAO1 strain and QSSMs (20 µM) for 20 hours and the expression of cell adhesion and inflammatory markers was assessed by flow cytometry (Gallios, Beckman-Coulter) using 1 × 105 endothelial cells stained
Protocol tips
Endothelial cells were detached using trypsin (Sigma-Aldrich, USA) and washed in PBS. Cells were then incubated with the primary antibodies at room temperature in the dark for 30 min. Further, the cells were washed twice and centrifuged at 400g, 10 min, in PBS suplemented with 1% BSA.
Downstream tips
Flow cytometry data were analyzed using the Gallios software (Beckman-Coulter).

Manufacturer protocol Publication protocol 1 Relevant paper

PE Mouse Anti-Human CD31 Clone L133.1

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
	Plasma samples were thawed at 37o C and centrifuged at 17,000 g for 10 min at RT. Pelleted material was resuspended in AnnV-binding buffer according to manufacturer´s instructions and aliquoted into BD Truecount Tubes

Protocol tips
Plasma samples were thawed at 37o C and centrifuged at 17,000 g for 10 min at RT. Pelleted material was resuspended in AnnV-binding buffer according to manufacturer´s instructions and aliquoted into BD Truecount Tubes

Manufacturer protocol Publication protocol 1 Relevant paper

PE Mouse Anti-Human CD31

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
	MV in 50 μl aliquots were labeled.	quantified by FACS (FACSCanto™) with a size > 150 nm as described previously

Protocol tips
MV in 50 μl aliquots were labeled.
Downstream tips
quantified by FACS (FACSCanto™) with a size > 150 nm as described previously

Manufacturer protocol Publication protocol 1 Relevant paper

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