Flow cytometry Anti-bodies Mouse - CD146/MCAM

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 CD146

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
	Each cell type was single cell suspended and, incubated in FACS buffer containing pre-conjugated antibodies (see supplementary Table 2) for 20 min on ice. Following incubation, cells were washed twice with FACS buffer.	All the flow cytometry performed using a Cell Lab Quanta SCMPL. The data were analyzed using Kaluza®1.2 software (Beckman Coulter, Denmark) and the expression of each CD markers on the cells was calculated based on the percentage and mean fluorescence intensity (MFI).

Protocol tips
Each cell type was single cell suspended and, incubated in FACS buffer containing pre-conjugated antibodies (see supplementary Table 2) for 20 min on ice. Following incubation, cells were washed twice with FACS buffer.
Downstream tips
All the flow cytometry performed using a Cell Lab Quanta SCMPL. The data were analyzed using Kaluza®1.2 software (Beckman Coulter, Denmark) and the expression of each CD markers on the cells was calculated based on the percentage and mean fluorescence intensity (MFI).

Manufacturer protocol Publication protocol 1 Relevant paper

Alexa Fluor® 488 Rat Anti-Mouse CD146

BD Biosciences

Upstream tips	Protocol tips	Downstream tips
Spleens, lymph nodes, lungs, and livers were harvested for IL-17 analysis on d28, d42, and d56, and single cell suspensions were made. The cells were stimulated with Cell Stimulation Cocktail plus Protein Transport Inhibitors (eBioscience, 00-4975-93) for 5 hours at 37°C in complete-RPMI.	Cells were stained with fixable viability dye eF780 or eF506 (eBioscience). All cells were fixed and permeabilized by the FOXP3 Fixation/Permeabilization Concentrate and Diluent kit (eBioscience).

Upstream tips
Spleens, lymph nodes, lungs, and livers were harvested for IL-17 analysis on d28, d42, and d56, and single cell suspensions were made. The cells were stimulated with Cell Stimulation Cocktail plus Protein Transport Inhibitors (eBioscience, 00-4975-93) for 5 hours at 37°C in complete-RPMI.
Protocol tips
Cells were stained with fixable viability dye eF780 or eF506 (eBioscience). All cells were fixed and permeabilized by the FOXP3 Fixation/Permeabilization Concentrate and Diluent kit (eBioscience).

Manufacturer protocol Publication protocol 1 Relevant paper

PE anti-mouse CD146 Antibody

BioLegend

Upstream tips	Protocol tips	Downstream tips
	Adherent endothelial cells were detached from culture flasks with trypsin/EDTA (Life Technologies), washed and resuspended in PBS (Gibco Life Technologies) containing 1% FCS. Cells (106 cells/ml) were then incubated with respective FITC-, PE- or APC-conjugated monoclonal antibodies for 20 min at 4°C. Fluorescence antibody-labeled cells were then washed twice in cold PBS with 1% FCS and analyzed using a flow cytometer (BD Bioscience; San Jose, CA, USA).

Protocol tips
Adherent endothelial cells were detached from culture flasks with trypsin/EDTA (Life Technologies), washed and resuspended in PBS (Gibco Life Technologies) containing 1% FCS. Cells (106 cells/ml) were then incubated with respective FITC-, PE- or APC-conjugated monoclonal antibodies for 20 min at 4°C. Fluorescence antibody-labeled cells were then washed twice in cold PBS with 1% FCS and analyzed using a flow cytometer (BD Bioscience; San Jose, CA, USA).

Protocol tips

Adherent endothelial cells were detached from culture flasks with trypsin/EDTA (Life Technologies), washed and resuspended in PBS (Gibco Life Technologies) containing 1% FCS. Cells (106 cells/ml) were then incubated with respective FITC-, PE- or APC-conjugated monoclonal antibodies for 20 min at 4°C. Fluorescence antibody-labeled cells were then washed twice in cold PBS with 1% FCS and analyzed using a flow cytometer (BD Bioscience; San Jose, CA, USA).

Manufacturer protocol Publication protocol 1 Relevant paper

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