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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 new MICS (multiparameter imaging cell screen) 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.
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