Flow cytometry Anti-bodies Human - Vimentin

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

Anti-human vimentin (clone V9)

ARA Antibodies for Research Applications B.V.

Upstream tips	Protocol tips	Downstream tips
	One million cells were incubated with 100 μl of MAb mixture directed against vimentin (clone V9-2b, dilution 1:5 culture supernatant (Antibodies for Research Applications, Gouda, Netherlands) overnight at 4°C. Cells were washed and incubated with 100 μl of premixed subclass-specific secondary Ab. DNA was labelled with PI and RNase (Sigma-Aldrich) treated (analyzed using a FACSCalibur flow cytometer, BD Biosciences, San Jose, CA) or DAPI (analyzed using an LSRII flow cytometer BD Biosciences).	A minimum of 30,000 single cell events were collected for each sample.

Protocol tips
One million cells were incubated with 100 μl of MAb mixture directed against vimentin (clone V9-2b, dilution 1:5 culture supernatant (Antibodies for Research Applications, Gouda, Netherlands) overnight at 4°C. Cells were washed and incubated with 100 μl of premixed subclass-specific secondary Ab. DNA was labelled with PI and RNase (Sigma-Aldrich) treated (analyzed using a FACSCalibur flow cytometer, BD Biosciences, San Jose, CA) or DAPI (analyzed using an LSRII flow cytometer BD Biosciences).
Downstream tips
A minimum of 30,000 single cell events were collected for each sample.

Publication protocol 1 Relevant paper

Anti-Vimentin antibody (ab137321)

Abcam

Upstream tips	Protocol tips	Downstream tips
	hPDLCs were rinsed with PBS for 3 times and fixed in 4% paraformaldehyde for 20 min. The fixed cells were treated with 0.5% Triton X-100 and 3% hydrogen peroxide for 15 min. The cells were then incubated for 1 h at 37 °C with rabbit anti-vimentin antibody (1:500).	Thereafter, the cells were washed with PBS for 3 times and incubated with corresponding secondary antibodies for 30 min at 37 °C. Finally, the cells were stained with a DAB kit (ZSGB-BIO, Beijing, China) and haematoxylin and viewed under an inverted microscope (Olympus, IX71, Tokyo, Japan).

Protocol tips
hPDLCs were rinsed with PBS for 3 times and fixed in 4% paraformaldehyde for 20 min. The fixed cells were treated with 0.5% Triton X-100 and 3% hydrogen peroxide for 15 min. The cells were then incubated for 1 h at 37 °C with rabbit anti-vimentin antibody (1:500).
Downstream tips
Thereafter, the cells were washed with PBS for 3 times and incubated with corresponding secondary antibodies for 30 min at 37 °C. Finally, the cells were stained with a DAB kit (ZSGB-BIO, Beijing, China) and haematoxylin and viewed under an inverted microscope (Olympus, IX71, Tokyo, Japan).

Manufacturer protocol Publication protocol 1 Relevant paper

Recombinant Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547)

Abcam

Upstream tips	Protocol tips	Downstream tips
The cells were treated with 0.25% trypsin (containing 0.02% EDTA) to produce a single cell suspension, followed by centrifugation at 1,000 × g for 5 min, and the supernatant was discarded. After washing in PBS, the cells were centrifuged and resuspended and then fixed with 95% ethanol.	Vimentin Ab 1:25; 40 µl at 4°C for 20 h)

Upstream tips
The cells were treated with 0.25% trypsin (containing 0.02% EDTA) to produce a single cell suspension, followed by centrifugation at 1,000 × g for 5 min, and the supernatant was discarded. After washing in PBS, the cells were centrifuged and resuspended and then fixed with 95% ethanol.
Protocol tips
Vimentin Ab 1:25; 40 µl at 4°C for 20 h)

Manufacturer protocol Publication protocol 1 Relevant paper

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