Flow cytometry Anti-bodies Mouse - CD73

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

CD73 Monoclonal Antibody (AD2), PE, eBioscience™

eBioscience

Upstream tips	Protocol tips	Downstream tips
Lungs were perfused with 10 ml PBS. A single-cell suspension was obtained by using C tubes (Miltenyi Biotec), followed by incubation with 1 mg/ml of collagenase D (Roche) and 0.1 mg/ml of DNase (AplliChem) for 1 h at 37°C. Cells were washed and suspended in PBS containing 3% FBS. Erythrocytes were lysed in ammonium-chloride-potassium lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA, H2O), and cells were washed with PBS.	Finally, cells were incubated with Fc-Block antibody (anti-CD16/CD32; BD Biosciences) for 15 min at 4°C	Cells were analyzed with a MACSQuant cytometer system (Miltenyi Biotec) and with FlowJo software (Treestar).

Upstream tips
Lungs were perfused with 10 ml PBS. A single-cell suspension was obtained by using C tubes (Miltenyi Biotec), followed by incubation with 1 mg/ml of collagenase D (Roche) and 0.1 mg/ml of DNase (AplliChem) for 1 h at 37°C. Cells were washed and suspended in PBS containing 3% FBS. Erythrocytes were lysed in ammonium-chloride-potassium lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA, H2O), and cells were washed with PBS.
Protocol tips
Finally, cells were incubated with Fc-Block antibody (anti-CD16/CD32; BD Biosciences) for 15 min at 4°C
Downstream tips
Cells were analyzed with a MACSQuant cytometer system (Miltenyi Biotec) and with FlowJo software (Treestar).

Manufacturer protocol Publication protocol 1 Relevant paper

PE Rat Anti-Mouse CD73

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

CD73 Monoclonal Antibody (AD2), APC, eBioscience™

eBioscience

Upstream tips	Protocol tips	Downstream tips
	Cells were resuspended in flow cytometry staining buffer, incubated with anti-mouse CD16/CD32 antibodies to block FcR, and then subjected to staining with different combinations of cell surface markers and CD73 and analyzed by flow cytometry.

Protocol tips
Cells were resuspended in flow cytometry staining buffer, incubated with anti-mouse CD16/CD32 antibodies to block FcR, and then subjected to staining with different combinations of cell surface markers and CD73 and analyzed by flow cytometry.

Manufacturer protocol Publication protocol 1 Relevant paper

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