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Global 5mC Detection by Flow Cytometry


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#1 SelcenC

SelcenC

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Posted 16 February 2010 - 09:15 PM

Hi,

First I will ask a question straigth away without stealing time of some of you who are not interested in this topic. :D

Does anyone use flow cytometry for detection of Global DNA Methylcytosine???

If yes or somehow interested, keep reading please?

I have been trying to optimize of global DNA methyl cytosine staining for flow cyto. The protocol i am revising for flow has been used for immunostanining of 5mC. (The last version of protocol is attached.)

1. My stable problem is very high or close noise to signal OR less signal then usual. I have checked the working efficiency of antibodies yesterday (by immunofluorescence), they worked ok. In theory my washing steps are organized fine.

2. Also when I add HCL to cells in suspension. Cells become stuck suddenly and then be harder to be dissolved that makes me think that it causes less antibody binding (just outside cells of aggregation). For this acid step do you have any suggestions?

So please give your advises and share your well working protocol for that,


Thank you so much

Selcen

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Edited by Selcen Celik, 16 February 2010 - 09:17 PM.

- Selcen -





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