Isolation of CD4+/CD8+ TIL subpopulation with REAlease Technology followed by dead cell removal improves cell vitality
Using the REAlease CD4/CD8 (TIL) MicroBead Kit, human directly on dissociated tumor tissue provides highly pure CD4+/CD8+ human TILs and delivers MicroBead- and label-free cells. Isolated cells are suitable for further processing, e.g., with the MicroBead-based Dead Cell Removal Kit, human, thereby improving the vitality of CD4+/CD8+ TILs from 80% to nearly 90%.
One major goal in immunotherapies is to understand how the composition of tumor-infiltrating myeloid and lymphoid cells contributes to patient stratification. However, TIL numbers can be very low and small subpopulations might be lost in background noise, particularly in single-cell analyses. Therefore, serial sorting of TIL subpopulations is highly advantageous in order to investigate the immune cell composition.
Background-free flow analysis of TILs
Using hybridoma-derived antibodies to identify TILs leads to a gross overestimation of the frequency of immune cell subpopulations present in the tumor. This experimental artifact is most likely caused by unspecific binding of hybridoma-derived antibodies to FcγRs on immune cells, as it is significantly reduced when using an FcR blocking reagent.
In contrast, assessment of TIL frequency using REAfinity™ Recombinant Antibodies is unchanged in the presence or absence of FcR blocking, providing a more exact analysis of respective cell populations, even in the absence of FcR blocking.
REAfinity Antibodies are highly specific recombinant antibodies that provide superior lot-to-lot consistency and purity compared to mouse or rat hybridoma-derived monoclonal antibodies.
|Panel 1||Clone||Fluorochrome||Panel 2||Clone||Fluorochrome|
|Lag-3||REA776||VioBright™ 515||CD103||REA789||VioBright 515 |
|CD4||REA604||PE-Vio® 615||CD4||REA604||PE-Vio 615|
|CD44||REA664||APC-Vio 770||CD44||REA664||APC-Vio 770|
The tumor microenvironment (TME) is inherently complex due to the highly variable cell composition as well as to interactions of thousands of proteins involved in metastasis formation. Thus, an in-depth analysis of tumor tissues requires the examination of a plethora of parameters in order to decipher the underlying principles. Currently available techniques can provide only a very limited perspective on TME complexity.
Miltenyi Biotec’s MICS (MACSimaTM Imaging Cyclic Staining) technology impressively overcomes these limits as it allows the fluorescence microscopic analysis of hundreds of markers on a single sample, without any harm, in a fully automated manner.