Flow cytometry Anti-bodies Mouse - CD137

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

CD137 (4-1BB) Monoclonal Antibody (17B5), APC, eBioscience™

eBioscience

Upstream tips	Protocol tips	Downstream tips
Following the isolation of liver metastasis pancreatic tumor infiltrating immune cells, the cells from mice from the same treatment group were pooled and resuspended in PBS at a concentration of 2 × 107 cells/mL.	For each treatment group, triplicates of this pooled cell suspension were placed into wells of a 96-well plate at a volume of 100 μL per well. The cells in each well were stained with Live-Dead Aqua (Invitrogen) for 30 min on ice, washed twice with PBS and then blocked with rat anti-mouse Fc antibody (CD16/CD32, clone 2.4G2, BD Biosciences) in FACs buffer for 10 min on ice. The FACs buffer consisted of HBSS (Sigma) with 2% bovine calf serum (Sigma), 0.1% sodium azide (Sigma) and 0.1% HEPES. After Fc blocking, the cells were stained for the following anti-mouse fluorophores for 1 h on ice.	The cells were then washed twice and resuspended in FACs buffer, and flow cytometry was performed using the Gallios flow cytometer (Beckman Coulter).

Upstream tips
Following the isolation of liver metastasis pancreatic tumor infiltrating immune cells, the cells from mice from the same treatment group were pooled and resuspended in PBS at a concentration of 2 × 107 cells/mL.
Protocol tips
For each treatment group, triplicates of this pooled cell suspension were placed into wells of a 96-well plate at a volume of 100 μL per well. The cells in each well were stained with Live-Dead Aqua (Invitrogen) for 30 min on ice, washed twice with PBS and then blocked with rat anti-mouse Fc antibody (CD16/CD32, clone 2.4G2, BD Biosciences) in FACs buffer for 10 min on ice. The FACs buffer consisted of HBSS (Sigma) with 2% bovine calf serum (Sigma), 0.1% sodium azide (Sigma) and 0.1% HEPES. After Fc blocking, the cells were stained for the following anti-mouse fluorophores for 1 h on ice.
Downstream tips
The cells were then washed twice and resuspended in FACs buffer, and flow cytometry was performed using the Gallios flow cytometer (Beckman Coulter).

Manufacturer protocol Publication protocol 1 Relevant paper

CD137 (4-1BB) Monoclonal Antibody (17B5), PE, eBioscience™

eBioscience

Upstream tips	Protocol tips	Downstream tips
The flow cytometry was performed to observe the CD137 level of membrane.	Cells were digested and washed with PBS. Anti-CD137-PE (eBioscience, USA) was diluted according to the protocol and was added to 100 μl cell suspension for 30 min at 4°C. After the incubation, the cells were washed one time and then analyzed by flow cytometry (BD Canto).

Upstream tips
The flow cytometry was performed to observe the CD137 level of membrane.
Protocol tips
Cells were digested and washed with PBS. Anti-CD137-PE (eBioscience, USA) was diluted according to the protocol and was added to 100 μl cell suspension for 30 min at 4°C. After the incubation, the cells were washed one time and then analyzed by flow cytometry (BD Canto).

Manufacturer protocol Publication protocol 1 Relevant paper

CD137 (4-1BB) Monoclonal Antibody (17B5), eFluor 450, eBioscience™

eBioscience

Upstream tips	Protocol tips	Downstream tips
	The cells were washed twice with PBS, resuspended and incubated in Fixation/Permeabilization buffer (eBioscience) for 30 min at 4 °C.

Protocol tips
The cells were washed twice with PBS, resuspended and incubated in Fixation/Permeabilization buffer (eBioscience) for 30 min at 4 °C.

Manufacturer protocol Publication protocol 1 Relevant paper

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