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Secondary Conjugate Protocol

Q: I’m using your secondary conjugate Mab-ZAP (Cat. #IT-04) and it’s not killing my cells. I’m not following the protocol on the data sheet. I’m doing flow cytometry. I have 70,000 cells per well. I mix Mab-ZAP with my primary antibody and add it. When I count the cells, there is no decrease. My cells grow very slowly.  I didn’t see anything after 72 hours.

A: The protocol on the data sheet is described in detail in the article by Kohls et al.

70,000 cells per well is a lot of cells per well. We use between 500 and 2500 over a 72-hour period and then develop with MTS.

If your cells are slow-growing, you may want to wait a little longer to develop the assay, because the whole metabolism process is slowed. This is a weakness of the MTS system — you have to have a certain number of cells in the end in the control cells to get a decent reading on your plate reader. 

In this case, you might want to try a more sensitive assay such as protein or DNA synthesis inhibition with incorporation of radiolabeled leucine or thymidine.

Related: ZAP Conjugates

References

  1. Kohls MD et al. Mab-ZAP: A tool for evaluating antibody efficacy for use in an immunotoxin. BioTechniques 28(1):162-165, 2000.

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