Flow cytometry Anti-bodies Human - FOXP3

Flow cytometry is an immunophenotyping technique whereby sing cell suspensions are stained for either cell surface markers or intracellular proteins by fluorescently-labelled antibodies and analyzed with a flow cytometer, where fluorescently-labelled molecules are excited by the laser to emit light at varying wavelengths, which is then detected by the instrument. There are several key criteria which are required to be kept in mind while designing a flow experiment- 1. Antibody titration (optimal dilution of antibodies should be calculated in order to avoid over- or under- saturated signals for proper detection of surface and intracellular markers), 2. Precision (3 or more replicates of the sample should be used per experiment), 3. Specificity (proper isotype controls should be included in the experiment), 4. Day-to-day variability (experiments should be repeated 3 or more times to ensure consistency and avoid variability due to flow cytometer settings), 5. Antibody interaction (Fluorescence minus one or FMO should be used, which is the comparison of signals from panel minus one antibody vs. the full panel), and 6. Antibody stability (fluorescently-labelled antibodies should be stored at 4C).

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Found 3 matching solutions for this experiment

FOXP3 Monoclonal Antibody (PCH101), PE, eBioscience™

eBioscience

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.

Manufacturer protocol Publication protocol 1 Relevant paper

FOXP3 Monoclonal Antibody (PCH101), Alexa Fluor 700, eBioscience™

eBioscience

Upstream tips	Protocol tips	Downstream tips
	Cell surface antibody staining was performed in PBS/0.5 % BSA/0.02 % sodium azide (PBA) buffer for 30 min at 4 °C.	. Stained cells were acquired on a LSR Fortessa (BD) and analyzed using DIVA software version 6.2. Events collected were generally >200,000 per sample, except for one tumor-infiltrating lymphocyte (TIL) sample (~35,000 cells). In the latter, still adequate numbers (~400) of Tregs could be detected.

Protocol tips
Cell surface antibody staining was performed in PBS/0.5 % BSA/0.02 % sodium azide (PBA) buffer for 30 min at 4 °C.
Downstream tips
. Stained cells were acquired on a LSR Fortessa (BD) and analyzed using DIVA software version 6.2. Events collected were generally >200,000 per sample, except for one tumor-infiltrating lymphocyte (TIL) sample (~35,000 cells). In the latter, still adequate numbers (~400) of Tregs could be detected.

Manufacturer protocol Publication protocol 1 Relevant paper

PE-CF594 Mouse Anti-Human FoxP3

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
	Cell surface antibody staining was performed in PBS/0.5 % BSA/0.02 % sodium azide (PBA) buffer for 30 min at 4 °C.	Stained cells were acquired on a LSR Fortessa (BD) and analyzed using DIVA software version 6.2. Events collected were generally >200,000 per sample, except for one tumor-infiltrating lymphocyte (TIL) sample (~35,000 cells). In the latter, still adequate numbers (~400) of Tregs could be detected.

Protocol tips
Cell surface antibody staining was performed in PBS/0.5 % BSA/0.02 % sodium azide (PBA) buffer for 30 min at 4 °C.
Downstream tips
Stained cells were acquired on a LSR Fortessa (BD) and analyzed using DIVA software version 6.2. Events collected were generally >200,000 per sample, except for one tumor-infiltrating lymphocyte (TIL) sample (~35,000 cells). In the latter, still adequate numbers (~400) of Tregs could be detected.

Publication protocol 1 Relevant paper

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