I need to analyze necrosis and apoptosis and my idea is to stain the spheroids with FITC Annexin V and propidium iodide and analyse by flow cytometry. Does anyone have experience with this technique? Can anyone suggest a protocol?
Annexin V and PI staining works very well. I recommend a conjugated Annexin V from Invitrogen (choose your conjugate according to the filter available on your flow cytometer). Remember to collect all the cells, including the supernatant. (contains dead cells). Wash with PBS1X, trypisinize, resuspend in annexin binding buffer (see manufacturer instructions), count the cells, stain a fix amount of cells with Annexin +PI for 15min, and then sort on Flow. Two important consideration, include a negative control (no apoptosis) and a positive control (apoptosis)!
Annexin V and PI staining works very well. However, you have to digest the Spheroids into single cells with accutase or trypsin.
Thank you all. Xiaowen, how can I do it? Is the same protocol for monolayer?
You can use annexin v and PI for spheriod using with FACS.
Below reference may provides the protocol.
http://www.translational-medicine.com/content/4/1/12
For having good results you may dissociate the spheroid cell population before passing the cells through the flow cytometer. After that all the techniques for usual cell analysis are possible. the Annexin V/PI or Anexin V/7AAD staining will only allow the measurement of dead cells, so you may have to do other staining procedure like caspase activity detection, ROS and mitochondrial membrane potential measurement, calcium and K+ levels. If you have a UV laser you can use the NADPH fluorescence which will provide you an access to the redox status of you cells etc... Have fun with that.
Patrice
if with "spheroids" you mean shperes used in Stem cell research, you should dissociate them and stain , no suggestion if you want to keep the spheroids intact except classical immunofluorescence..Fo necrosis/apoptosis there is a nice kit from ENZO life science that is simple and helpful. Basically I usually dissociate the spheres (mechanical dissociation by micro pipet) and then stain them in FACS tubes according to the protocol. FOr AnnexinV and PI only, I suggest you to stain one sample only with PI and one only with Annexin for compensation. If you buy a commercial kit for annexinV PI just saty with the protocol. Mechanical dissociation should affect cell viability, if you have problems in dissociating spheres you could use TRIPLE buffer, broadly used in Stem cell research to dissociate spheres.
Thank you all. My ideia is to analyze the spheroids without dissociate them. I'll try confocal microscocopy. Do you know if is possible to use AnnexinV and PI fot confocal analyse?
I know this team is working with flow cytometry of whole spheroids (link is to their poster): http://3dbiomatrix.com/wp-content/uploads/2012/07/cyto2012-poster.jpg
I'm not sure of the final details of the work, but you can always contact them or the company making the flow cytometer. I hope this is helpful!
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