Thermo Fisher Scientific
a-mouse PD-L1 PerCP-eF710
46-5982-82
MIH5
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I tested this antibody to identify co-inhibitory molecules and their ligands on T cells in a large panel which also analyses B cells, macrophages, monocytes, DCs and NK cells. I used this antibody on splenocytes obtained from C57BL/6J mice infected with Plasmodium berghei ANKA (PbA), a mouse model to investigate malaria. CD274 (PD-l1) is a ligand to PD-1, perhaps the most known co-inhibitory molecules. Accordingly, this molecules was highly relevant to the panel. I aimed to analyse its expression on antigen-presenting cells, however, I was also able to detect PD-L1 expression on T cells.
Flow Cytometry
C57BL/6 splenocytes
1:100, 20 min, 4°C, in 50ul, in the dark
Fc Block
Fix/Perm buffer, 20 min, 4°C, in 70ul, in the dark
20 min, 4°C, in the dark, in 50µl Perm/wash
Flow cytometry
PD-L1 may be expressed on multiple cell types, including antigen-presenting cells as well as T cells. I used this antibody for a 41-color panel measured on a 5L Cytek Aurora. I preferred PerCP-eF710 over other PerCP-conjugates as its emission shifts further in the red emission range, away from similar emitting fluorochromes used in this panel (Ly-6G PerCP; PE-Cy5 CD127). Furthermore, brightness was sufficient to detect a clear shift in signal between naïve and PbA-infected animals. Shown here are monocytes (CD11b+ F4/80+) isolated from the spleens of C57BL/6 mice. Histogram shows cells from naïve (blue) and infected (red) animals. At a concentration of 1:100, this antibody can be used cost-effectively.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 Cytek Aurora.
N/A
Works well, even on a PerCp-based conjugate.
Was very happy with this antibody, which allowed me to utilize the emission range around 700nm on the blue laser line. Usually, these fluorochromes can cause issues.