Protocol tips
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Cells (106) were harvested after trypsinization, washed twice (for cell surface markers expression) or fixed with ethanol (for Ki-67 staining) and suspended in 500 μl PBS 1X containing 0,5% bovine serum albumin. For Ki-67 determination an allophycocyanin-conjugated mouse anti-human Ki-67 (Biolegend) and its respective isotype control APC Mouse IgG1, κ isotype Ctrl (FC) were used. The different antibodies were incubated for 30 min and washed to remove the excess of antibodies. |
The cytometric analysis was carried out using a fluorescence-activated cell sorting (FACS) Aria-II flow cytometer (BD Bioscience). The Flow Jo software was used for data acquisition and analysis, respectively, using measurements from 10,000 cells in each experiment. |
Protocol tips |
Cells (106) were harvested after trypsinization, washed twice (for cell surface markers expression) or fixed with ethanol (for Ki-67 staining) and suspended in 500 μl PBS 1X containing 0,5% bovine serum albumin. For Ki-67 determination an allophycocyanin-conjugated mouse anti-human Ki-67 (Biolegend) and its respective isotype control APC Mouse IgG1, κ isotype Ctrl (FC) were used. The different antibodies were incubated for 30 min and washed to remove the excess of antibodies. |
Downstream tips |
The cytometric analysis was carried out using a fluorescence-activated cell sorting (FACS) Aria-II flow cytometer (BD Bioscience). The Flow Jo software was used for data acquisition and analysis, respectively, using measurements from 10,000 cells in each experiment. |
Publication protocol
Cells (106) were harvested after trypsinization, washed twice (for cell surface markers expression) or fixed with ethanol (for Ki-67 staining) and suspended in 500 μl PBS 1X containing 0,5% bovine serum albumin. Cells were stained with FITC-conjugated mouse anti-human CD44 and R-Phycoerythrin-conjugated mouse anti-human CD24 (both from Invitrogen). Cells incubated without antibody were used as a blank. For Ki-67 determination an allophycocyanin-conjugated mouse anti-human Ki-67 (Biolegend) and its respective isotype control APC Mouse IgG1, κ isotype Ctrl (FC) were used. The different antibodies were incubated for 30 min and washed to remove the excess of antibodies. The cytometric analysis was carried out using a fluorescence-activated cell sorting (FACS) Aria-II flow cytometer (BD Bioscience). The Flow Jo software was used for data acquisition and analysis, respectively, using measurements from 10,000 cells in each experiment.
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