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a-mouse PD-1 APC
109112
RPM1-30
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I wanted to study T cell exhaustion/ regulation in mice during the infection with Plasmodium berghei ANKA, a murine model for malaria. I used this antibody in multiple organs (Spleen, liver, brain, ling, blood) and it worked well.
Flow Cytometry
C57BL/6 splenocytes
1:100 Surface staining, 20 min, 4°C, in 50ul, in the dark
Fc Block
Fix/Perm buffer, 20 min, 4°C, in 70ul, in the dark
Intracelluar staining 20 min, 4°C, in the dark, in 50µl Perm/wash
Flow cytomtery
PD-1 is one of the key markers in T cell exhaustion, but sometimes difficult to stain. Accordingly, a bright fluorochrome, like APC, may be beneficial to detect a decent signal. With this antibody/ fluorochrome combination, I was able to detect strong PD-1 expression, almost separating a distinct population (rather than a “smear”) of PD-1 positive T cells. PD-1 expression correlated with other co-inhibitory molecules (LAG-3, TIM-3,…) in my model. In the following, you will find my staining protocol in short. Incubation steps were performed at 4°C in the dark. 1.Count and adjust splenocytes to 1.5E6 cells, plate in a 96-well plate. 2.If required, stain cells with viability dye. Wash. 3. Stain with extracellular master mix for 20 min. Wash 2x. 4. Fixate/ Permeabilize with buffer of choice, depending on application. I used ThermoFisher FoxP3 fixation/permeabilization kit for 20 min. Wash with perm/ Wash 2x. 5. Perform intracellular staining for 20 min. Wash 2x. 6. Resuspend in FACS buffer and acquire as soon as possible. Here, data was acquired on a 5L 5 L BD LSRFortessa with the appropriate filters.
N/A
Cheap antibody, bright signal.
I would use this antibody again, especially on flow cytometers with limited lasers/ detectors.