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PE/Cy7 anti-human CD45RA Antibody
304126
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The expression of CD45RA on T cell serves to identify distinct subsets. Usually CD45RA is expressed on naive T cells along with the expression of CCR7 and CD62L. These T cell lack the expression of CD45RO. The naive phenotype can be assessed by looking into a subset which is CD45RA+ CD45RO- CCR7+ and CD62L+. Interestingly, CD45RA is also expressed on effector T cells which lacks CCR7 and CD62L expression. The phenotype for effector T cell is thus considered to be CD45RA+ CD45RO- CCR7- CD62L- and is again a different subset from T effector memory.To analyze these T cell subset, it is important to gate on CD45RA+ cells first and then check expression of other receptor molecules withing that gate. Using multi-color flow cytometric experiment with all the mentioned surface markers allows to determine distinct T cells naive/memory/effector subsets.
Flow Cytometry
Human PBMC or whole blood
Human PBMC ( or 100ul of whole blood) were stained with surface antibodies for CD4/CD8/ CD45RA/ CD45RO/ CCR7/ CD62L. Antibodies used were FITC anti-CD4, PE anti-CD8, PercpCy5.5 anti-CD62L, PEcy7 anti-CD45RA, APC anti-CD45RO and APC cy7 anti-CCR7. Cells were stained for 30 mins in dark and washed twice with staining buffer( RBC lysis buffer was used if the starting material was whole blood). Cells were re-suspended in 500ul of staining buffer and acquired in 6 color flow cytometry. Analysis was done in a manner that CD4 and CD8 T cells were gated first. Then on that gate, the expression of CD45RA was analyzed and the CD45RA+ cells were again gated out. Next the expression of other surface markers were evaluated to scrutinize different T cell subset.
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Flow cytometric detection
There was a distinct subset of T c ells which expressed CD45RA. When plotted against isotype control, there was two distinct peaks for CD45RA positive and CD45RA negative population. The CD45RA + population was further gated to analyzed different subsets.
When analyzing different sub-population, the order of gating is important
Reliable and easy method of flow cytometric analysis of CD45RA expression
Nothing to mention
The bright staining of PECy7 anti-CD45RA antibody makes it easy to detect the cells which are positively stained.