FITC Mouse Anti-Ki-67 Set

Protocol tips

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.

Publication protocol

Extra‐ and intracellular immunofluorescence stainings of T cells were conducted using the following specific primary mouse anti‐human mAbs: FITC‐conjugated anti‐CD4 (mIgG2b, OKT4); Alexa Fluor488‐conjugated anti‐CD127 (mIgG1, HCD127); PE‐conjugated anti‐CD49d (mIgG1, 9F10); PerCP/Cy5.5‐conjugated anti‐CD127 (mIgG1, HCD127), ‐CD49d (mIgG1, 9F10), ‐CD4 (mIgG2b, OKT4); ‐LAP (TGF‐ß1, mIgG1, TW4‐2F8); APC‐conjugated anti‐CD25 (mIgG2b, 4E3), ‐CD69 (mIgG1, FN50), all obtained from BioLegend (Fell, Germany); PE‐conjugated anti‐CD25 (IgG2b, 4E3), biotin‐conjugated anti‐CD49d and ‐CD127, all from Miltenyi Biotec GmbH; FITC‐conjugated anti‐Ki‐67 (mIgG1, 35/Ki‐67, BD Bioscience, Heidelberg, Germany) and human/non‐human primate regulatory T cell staining kit 1 (eBioscience, Frankfurt, Germany), containing PE‐conjugated anti‐FoxP3 (ratIgG2a, PCH101), ‐ratIgG2a, FITC‐conjugated anti‐CD4 (OKT4) and APC‐conjugated anti‐CD25 (BC96). Control staining was performed with corresponding FITC‐, PE‐, Streptavidin‐PerCP/Cy5.5‐ or biotin‐conjugated mIgG1 (MOCP‐21), PE‐ or biotin‐conjugated mIgG2a (MOCP‐173); FITC‐, or APC‐conjugated mIgG2b (MPC/11), all obtained from BioLegend and PE‐conjugated mIgG1 (P3.6.2.8.1, eBioscience) isotype control antibodies. In addition, Streptavidin‐PerCP/Cy5.5 (Biolegend) was used as secondary staining reagent. 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). In case of biotin‐conjugated mAbs, cells were incubated with Streptavidin‐PerCp/Cy5.5 at a final concentration of 1:100 for 15 min at 4 °C followed by a second washing step before fixation. 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.

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