Anti-human vimentin (clone V9)

Protocol tips

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

Flow cytometry was performed as previously described [18]. In short, cell suspensions were prepared from 2-3 dewaxed 60 μm FFPE material sections. One million cells were incubated with 100 μl of MAb mixture directed against keratin (clone MNF116, 2 μg/mL, DAKOCytomation, Glostrup, Denmark and clone AE1/AE3, 5 μg/mL, Chemicon International Inc, Temecula, CA, USA), and 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 FITC or APC-labelled secondary reagents (Southern Biotechnology Associates, Birmingham, AL, USA). 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. A data file contained all events, included aggregates and debris, and data were analyzed using the WinList 8.0 and Mod-Fit 4.1 software packages (Verity Software House, Inc, Topsham, ME, USA). The fraction of keratin-positive/vimenitin-negative (K+) and keratin-positive/vimentin-positive (V+) cells was calculated after gating on single cells in the DNA-Area versus DNA-Width (doublet discrimination module) dot plot.

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