Flow cytometry Anti-bodies Mouse - CD140/PDGFR-α

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 CD140A

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

CD140a (PDGFRA) Monoclonal Antibody (APA5), PE, eBioscience™

eBioscience

Upstream tips	Protocol tips	Downstream tips
	Mouse lung fibroblasts were plated onto 6-well plates (1×105/well) and incubated in RPMI1640 medium supplemented with 10% FBS at 37°C under 5% CO2 in humidified air. Cells (5×105) were collected and incubated for 30 minutes on ice with PE anti-mouse CD140a (PDGFR-α) antibody	Stained cells were analyzed by flow cytometry using BD LSRFortessa for acquisition and BD FACSDiva software 6.0 (BD Biosciences, San Jose, CA) for analyses

Protocol tips
Mouse lung fibroblasts were plated onto 6-well plates (1×105/well) and incubated in RPMI1640 medium supplemented with 10% FBS at 37°C under 5% CO2 in humidified air. Cells (5×105) were collected and incubated for 30 minutes on ice with PE anti-mouse CD140a (PDGFR-α) antibody
Downstream tips
Stained cells were analyzed by flow cytometry using BD LSRFortessa for acquisition and BD FACSDiva software 6.0 (BD Biosciences, San Jose, CA) for analyses

Manufacturer protocol Publication protocol 1 Relevant paper

APC anti-mouse CD140a Antibody

BioLegend

Upstream tips	Protocol tips	Downstream tips
	Five million cells per animal were blocked with 1 µg Fc-block/tube and stained with anti-mouse PDGFRα antibody (APC-conjugated, clone APA5; Biolegend, 10 µg/ml) for 20 min at 4°C.	For FACS analysis a FACSCanto II (BD Biosciences) was used. Results were visualized by the FlowJo package (Tree Star Inc., Ashland, OR, USA).

Protocol tips
Five million cells per animal were blocked with 1 µg Fc-block/tube and stained with anti-mouse PDGFRα antibody (APC-conjugated, clone APA5; Biolegend, 10 µg/ml) for 20 min at 4°C.
Downstream tips
For FACS analysis a FACSCanto II (BD Biosciences) was used. Results were visualized by the FlowJo package (Tree Star Inc., Ashland, OR, USA).

Manufacturer protocol Publication protocol 1 Relevant paper

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