We are trying to detect mouse Granzyme B in conditioned-media (CM) of CD8 cells from mice spleens by ELISA. However, our results are below the detection levels (62.5 pg/ml). When we are using this ELISA on organ lysates (e.g., tumors), naturally we can readily detect Granzyme B. We are using a R&D systems Mouse Granzyme B DuoSet ELISA - DY1865. We tried to concentrate the CM using adequate centricons but it did not help. We are wondering if it is acceptable and double to detect Granzyme B in the lysates of the CD8 cells. Alternatively, is it acceptable to detect Granzyme B mRNA levels?
Regards,
Ziv
We do intracellular staining for granzyme B. This gives you the frequency of positive cells and if you do MFI insight into the levels of granzyme B. We typically also do perforin intracellular staining at the same time. You will of course also want to do membrane phenotypic staining for effector cells.
Hi Ziv Raviv
I briefly wanted to reach out concerning your question.
For the above mentioned experiment check out Figure 4 in our paper for the results and the materials and methods section for details concerning antibodies and staining protocols. You will also find an optimized protocol for perforin staining in the paper.
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For highly sensitive detection of (human) granzyme B check out our I-O panel.
https://www.olink.com/products-services/target/immuno-oncology/
All the best & good luck with your experiments,
Michael
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