how to fix embryoid body (EB) by formaldehyde - (Aug/26/2008 )
As is known to all, formation of embryoid body (EB) is essential during in vitro differentiation of ES cells. I have to use such EB body to do ChIP assay. EB body is a clump of cells. I have no idea how to fix them. Should I disperse the EB body into single cell by trypsinization before fixation? Has anyone handled with EB body? Please give me some suggestions! Thank you, all guys!
Answer from Fabio, 1st author of Molecular Cell paper published in 2008. Molecular Cell 30, 1-12,2008
I tried both ways of formaldehyde fixing for EBs, i.e. with and without dissociation before formaldehyde addition. For histone modification and RNA polymerase II ChIP it did not seem to make a big difference. In the end I decided to do dissociation prior to fixation because for sonication it worked slightly better than without dissociation (less clumps). However I did not try any other transcription factors, so I can not say if this can influence localization. If your factor of interest is very dynamic, it might be better to directly fix EBs as trypsinization is quite stressful for the cells. I would be less concerned about uneven fixation since you can play around with fixation time and formaldehyde concentration to improve enrichments once the ChIP works. You might also try ChIP with different amounts of chromatin and antibody, for me this had a big impact on the enrichments, especially for transcription factor ChIP.
I guess I would try both ways and check the ChIP enrichment on a number of positive and negative controls and then use the protocol which looks better on your controls.