Flow cytometry Anti-bodies Mouse - CD25

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

PE Rat Anti-Mouse CD25

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
Spleens and tumors were excised from the 4T1 tumor-bearing mice. Single cell suspensions were produced by homogenizing the spleen samples, and the tumors were minced and subsequently digested with 500 U/mL collagenase type IV (Sigma) for 1 h at 37 °C with agitation. The resulting single cell suspensions were suspended in RPMI 1640 with 10% fetal calf serum (FCS, Gibco, England).	anti-CD25 PE (clone PC61, BD Biosciences) antibodies were used to stain the cell surface of regulatory T cells (Tregs). The cells were then fixed and permeabilized for intracellular staining.	). All staining reactions were performed in a final volume of 100 μl at 4 °C. Data were acquired using a FACS Calibur flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo v7.6.2 software (Tree Star Inc., Ashland, OR, USA).

Upstream tips
Spleens and tumors were excised from the 4T1 tumor-bearing mice. Single cell suspensions were produced by homogenizing the spleen samples, and the tumors were minced and subsequently digested with 500 U/mL collagenase type IV (Sigma) for 1 h at 37 °C with agitation. The resulting single cell suspensions were suspended in RPMI 1640 with 10% fetal calf serum (FCS, Gibco, England).
Protocol tips
anti-CD25 PE (clone PC61, BD Biosciences) antibodies were used to stain the cell surface of regulatory T cells (Tregs). The cells were then fixed and permeabilized for intracellular staining.
Downstream tips
). All staining reactions were performed in a final volume of 100 μl at 4 °C. Data were acquired using a FACS Calibur flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo v7.6.2 software (Tree Star Inc., Ashland, OR, USA).

Manufacturer protocol Publication protocol 1 Relevant paper

FITC anti-mouse CD25 Antibody

BioLegend

Upstream tips	Protocol tips	Downstream tips
Isolation of splenocytes was done by mashing the spleen through a 70-μm cell strainer. The cells were washed with phosphate-buffered saline and after spinning at 300g for 6 minutes, the cell pellet was resuspended in 1 mL lysis buffer for 3 minutes.	The cells were then fixed and permeabilized. Thereafter, the antibody cocktail (CD4, CD3, CD11b, Ly6G, CD69, CD25 and CD62L) was added and incubated at 4°C for an additional 20 minutes and then analyzed by flow cytometry.

Upstream tips
Isolation of splenocytes was done by mashing the spleen through a 70-μm cell strainer. The cells were washed with phosphate-buffered saline and after spinning at 300g for 6 minutes, the cell pellet was resuspended in 1 mL lysis buffer for 3 minutes.
Protocol tips
The cells were then fixed and permeabilized. Thereafter, the antibody cocktail (CD4, CD3, CD11b, Ly6G, CD69, CD25 and CD62L) was added and incubated at 4°C for an additional 20 minutes and then analyzed by flow cytometry.

Manufacturer protocol Publication protocol 1 Relevant paper

CD25 Monoclonal Antibody (PC61.5), APC, eBioscience™

eBioscience

Upstream tips	Protocol tips	Downstream tips
For Treg cell staining, we used anticoagulant in whole blood for 200 μL per tube, and after lysis by Red Blood Cell Lysis Buffer (Beijing Solarbio Science and Technology Co., Beijing, China), cells were resuspended in PBS.	mixing and incubation at room temperature in the dark for 30 min. After washing 2 times, cells were fixed and permeabilized, the we added 10 μL Foxp3-PE (PE anti-mouse Foxp3, eBioscience, USA), followed by incubation for 30 min at room temperature.

Upstream tips
For Treg cell staining, we used anticoagulant in whole blood for 200 μL per tube, and after lysis by Red Blood Cell Lysis Buffer (Beijing Solarbio Science and Technology Co., Beijing, China), cells were resuspended in PBS.
Protocol tips
mixing and incubation at room temperature in the dark for 30 min. After washing 2 times, cells were fixed and permeabilized, the we added 10 μL Foxp3-PE (PE anti-mouse Foxp3, eBioscience, USA), followed by incubation for 30 min at room temperature.

Manufacturer protocol Publication protocol 1 Relevant paper

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