No discussions found
Start your discussion
Share your thoughts or question with experts in your field
Start a discussion
Found 3 matching solutions for this experiment
| Upstream tips |
Protocol tips |
Downstream tips |
|
Staining of surface molecules was always performed by incubating cells with specific mAbs or the respective isotype control for 15 min at 4 °C in the dark and at a concentration as recommended by the manufacturer. Then, cells were washed with MACS‐buffer (see above) and fixed with 1% formaldehyde (BÜFA Chemicals GmbH&Co.KG, Hude, Germany). |
When combining extra‐cellular and intra‐cellular stainings, first the surface staining was performed. After the last washing step, cells were treated with freshly prepared fixation/permeabilization solution for 10 min at room temperature in the dark, washed with permeabilization buffer (both from eBioscience) and followed by incubation with fluorochrom‐conjugated specific mAbs diluted in permeabilization buffer for 10 min at room temperature in the dark. After washing with permeabilization buffer, cells were finally fixed with 1% formaldehyde. Flow cytometric analysis was always performed with a FACS Calibur (BD Biosciences, Heidelberg, Germany) equipped with an argon laser. |
| Protocol tips |
| Staining of surface molecules was always performed by incubating cells with specific mAbs or the respective isotype control for 15 min at 4 °C in the dark and at a concentration as recommended by the manufacturer. Then, cells were washed with MACS‐buffer (see above) and fixed with 1% formaldehyde (BÜFA Chemicals GmbH&Co.KG, Hude, Germany). |
| Downstream tips |
| When combining extra‐cellular and intra‐cellular stainings, first the surface staining was performed. After the last washing step, cells were treated with freshly prepared fixation/permeabilization solution for 10 min at room temperature in the dark, washed with permeabilization buffer (both from eBioscience) and followed by incubation with fluorochrom‐conjugated specific mAbs diluted in permeabilization buffer for 10 min at room temperature in the dark. After washing with permeabilization buffer, cells were finally fixed with 1% formaldehyde. Flow cytometric analysis was always performed with a FACS Calibur (BD Biosciences, Heidelberg, Germany) equipped with an argon laser. |
| 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. |
| Upstream tips |
Protocol tips |
Downstream tips |
|
Intracellular staining of Ki-67 was performed using a FoxP3 transcription factor staining buffer set (eBioscience) and specific antibodies. |
All stained samples were acquired with a LSR II flow cytometer (BD Biosciences), and the data were analyzed by FlowJo software version 10.4.0 (Treestar). |
| Protocol tips |
| Intracellular staining of Ki-67 was performed using a FoxP3 transcription factor staining buffer set (eBioscience) and specific antibodies. |
| Downstream tips |
| All stained samples were acquired with a LSR II flow cytometer (BD Biosciences), and the data were analyzed by FlowJo software version 10.4.0 (Treestar). |
Can't find the product you've used to perform this experiment? It would be great if you can help us by
Adding a product!