APC anti-human Ki-67 Antibody

Protocol tips

Upstream tips	Protocol tips	Downstream 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.	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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