How to show a good control for cell treatment - (Jul/27/2006 )

Hi,
I am working on HT-29 cell line, i.e a human colon cancer cell line. I am trying to see the effects of different phospholipase inhibitors on these cells,after treating them with arachidonic acid. My cell count is around 1 miillion/ml. and I am using 25 um Arachidonic acid, Free AA is supposed to induce apoptosis.The PLA2 inhibitors blocks the activity of different phospholipases like(sPLA2,cPLA2 and iPLA2), we are interested to see the effect of these 3 PLA2 s. I am trying to analyse my experiment with the help of flow cytometry, after incubating my cells for 2 hours. The sad part is that I could not be able to see a good control, I have done this experiment for 5 times,and hardly 10 % cells were alive after 2 hours. Untill I get a good control I could not show any data whatever I get from the other factors.
I am using Mckoy's 5A media, which is specific for this cells, as suggested by ATCC.This media consists 10%FBS, is there any effect of FBS on the death of cells? The cells became apoptotic automatically as the Flow data shows.
Please suggest me something.

Deb

Have u observed the cells after adding the inhibitors without using flow cytometry. R the cells alive?

U have to increase the number of cells alive, as 10% is too less to show much. U have to titrate ur arachidonic acid conc. or the inhibitor conc.

Can you also consider using "live/death staining kit". It is fast and has less steps involved than FACS.

I have all the controls as mentioned, I am measuring the apoptosis by using Annexin V and necrotic cells by Propidium iodide. My desired result after adding the inhibitors is to see the change in the rate of apoptosis. PLA2 helps in the conversion of AA-PL molecule into free arachidonic acid, out of which some arachidonic acid are converted into inflammatory molecules by COX-2 and LOX, and the rest Free Arachidonic acid induces the apoptosis. So, blocking the PLA2 is important to see the change in apoptosis, as well as to see the interaction between different PLA2 s.
I am anxious to improve my control cells, I dont know whether the cell line is itself kind of apoptotic or not.
Whats is LIVE/DEATH STAINING KIT?I dont know about this..Could you please tell me about this?

Thanks to all for the replies. Looking forward for more suggestion.

Deb

After 2 hours of my treatment I take out the media, keep it in a FACS tube, then I detach the cells using FACS buffer, then adding Media for neutralizing. Then adding the cell suspension to FACS tube, and then spin it down to get the cell pellet. Removed the supernatent, added 100ul of FACS buffer, and then added Annexin V and PI, and incubated in room temp for 15 minutes.
After that I do the Flow cytometry assay.

Any suggestion???

Deb

[quote name='debarshir' date='Jul 28 2006, 04:34 PM' post='61673']
After 2 hours of my treatment I take out the media, keep it in a FACS tube, then I detach the cells using FACS buffer, then adding Media for neutralizing. Then adding the cell suspension to FACS tube, and then spin it down to get the cell pellet. Removed the supernatent, added 100ul of FACS buffer, and then added Annexin V and PI, and incubated in room temp for 15 minutes.
After that I do the Flow cytometry assay.

Any suggestion???

It sounds like you are doing it right. The reason why I asked is because Annexin V/PI (some peopel use 7 AAD instead of PI) assay is very sensitive and the accurate results depend on how intact the cell memabrane is . If you damage the cell membrane while manipulating the cells (or if you fix the cells), it may became permeable for PI or pertubate the PS residues and give you false results. How du you detach the cells?
When you look at your Forward vs. Side scatter flow data do you see any change in cell size, increase in cell debris... anything indicative of cell death ?

Deb,

I have used live/dead staining kit from Invitrogen/Mol Probes (Calcein AM/PI). Because on you hand cells got killed real fast, I thought you can use fluorescence microscopy instead of FACS to get results fast.

So you are using AA and PLA2 blockers as positive control. Is there a known agent that can activate PLAs for HT-29? Can you use that as positive control instead?

Also, AA is produced through PLA2 activation, do you beleive that there is a second wave of AA production stimulated by AA itself? Am I missing somthing here?

is there any fixative that can be used to fix cells after annexin V/PI staining. I will be doing a time course shortly but it will be very limited if i cannot freeze away samples