For single staining, if I undertood you right, you use histogram to analyse the changes in fluorescence. You can gate the poistive and negative populations and get your results as percentage of population or you can analyse the whole population with the changes in the mean fluorescence...

If you think you need a 2 value graphic (Dot plot) you can do it with the PI signal vs the FSC (forward scattering) since cell change their shape as they start to get necrotic.

BD has a nice intro paper into flow cytometry, it could help you little bit more

good luck

Hi Sanjaya,

A histogram for the single staining might be the best way to go for. Nevertheless, you can also do this dot plot of your color against the FSC as Luisa suggested. I can send you an e-mail with an example what you can draw on a FACS calibur and how it then should look like. Your PI positive cells will be the dead cells. Just to get a better result I would suggest to add the PI just before measurement in each individual sample. Do not stand it around to long because the dead cells will then take up more of the PI and your color is getting too strong (out of range). Furthermore, do not forget to measure first the unstained and make the setting with this and then the stained one. Include a positive control with roughly 50% dead cells, that you see how it should look like.

Good luck!

I can send you an e-mail with an example what you can draw on a FACS calibur and how it then should look like. ia this offer san be also to me as i am teaching flow cytometry to my post graduate students Thank you in advance

@Hoda, I do not have your e-mail address.

Thank you , my email [email protected]

Thank you Luisa and Nicole. I will try out the method hope this time everything goes well. If not then will again be asking you all here

Hello Luisa and Nicole,

I also found out that cell death can be analysed thru the cell cycle assay using the PI stain-by measuring the amount of cells in the sub-G1 phase. However, I guess that the techniques you mentioned above-the histogram aalysis method were for measuring the percentage of the cells that are stained PI positive as the cell death percentage. And that for carrying on the cell death analysis I do not need to fix my cells as required in cell cycle assays. Since I also received the details of carrying out the procedure and assaying cell death by the fixation method. I wanted to be sure that- the method of cell death assay can be done without fixation and FACS analysis carried on by the histogram method.

I actually wanted to post some pics of the result I was expecting- the protocol i wanted to follow from an article I read, seems Research gate needs to add that feature of allowing sharing of pics.

Hello Sanyaja,

the staining I mentioned is the one you expected without fixation and measuring the PI positive cells as cell death. Therefore, it is much easier but you need to measure your samples directly after harvesting.

I hope that helps!

can anybody share little GS monoclonal antibody

Hi! About cell cycle with PI... it works well with fixation, but BECAREFUL!!! For cell viability you cannot fix the cells.

The principle of PI is that it enters only those cell with damaged membranes and as soon as it binds with the DNA increases the fluorescence up to 30 times, that is why you can distinguish between dead (or "membrane- damaged") cells and live (membrane intact) cells. In the case of cell cycle, you can see only variation in the DNA content of the cells: For cancer cells you have to synchronize them, then you can evaluate if your cells are arrested in some of the cell cycle parts by your treatment. In that case, if fixation permeabilzed the cells, it is not a big deal, you just wanted to look at the arrest of the cells, or in general any cell cycle disruption/change. For viability you need them as natural posible, so you don't get false positive results.

Good luck

Thanks Nicole and Luisa for the detailed instruction. Guess I will proceed with it once this week hope everything goes fine this time.

Hello there,

I later on dropped the plan for single staining and followed the protocol for Annexin V-PI staining instead, but then still I had problems with the final analysis. I am not able to see the apoptotic or necrotic cells in the expected amounts.

It seems to me the problem is in setting up the baseline. We are using the control of untreated sample as a baseline..........which I am not sure should we used.......since control itself may have some population of dead cells and decreasing the FL1 and FL2 to a level to compress the whole spectra so that the control does not have apoptotic and necrotic cells should not be an option in my opinion....... and am looking for steps to be followed or setting up the parameters during the analysis.

The settings used were

Parameter Detector Voltage Amp Gain

P1 E1 4.62

P2 SSC 321

P3 FL1 190

P4 FL2 235

P5 FL3 551

Tried changing the Detector voltage to FL1 320 and FL2 380, which caused the dot plot to shift upward.

appeared to have very less viabile cells while doing it. Well I need to get to a balance point ......... in set up of flow cytometer

Compensation

FL1 2.4 P1/P2 Lin mode

FL2 35

P3/P4 Log mode

Sorry for troubling u all again.

The data is quite Raw in its form- I did not feel like manipulating it

Samples

1--Control

2,3&4-- cell death induced

5,6--Positive control drug added to offer resistance to cell death

7--Cell population induced to die by not changing medium for a long time--just wanted to check the state of cells when they are kept in low serum medium for 9 days of time.

http://img403.imageshack.us/img403/1048/30thnov.jpg

http://img403.imageshack.us/img403/1048/30thnov.jpg

Regards

Sanjaya

Dear Sanjaya...

I will write to you more extensively later (when I have some more time) but the first thing that I saw from your FSC vs SSC i that your FSC is set up to low, you need to increase the FSC and lower the SSC to see the different populations. It seems that your cells are small and highly granulated. Once your can see 2 populations clearly, you are on the right way. The population that will come up in the lower left (low FCS and low SSC) are the "standard" dead cells ( cells that got apoptotic and small... ugly =) ), This ones you can gate out so your data for the anexin will not show the "original" dead. The other option: Synchronize your cells... you get better data.... I hope it helps for now...

Greetings

Luisa

Thank you Luisa,

Yeah actually we had tried increasing both the FSC and SSC at the same time and the problem seems to have arised there, since the graph came just like extrapolating the thing we got instead of getting two populations clearly. I am trying with flow cytometry again tomorrow. Hope will have better luck tomorrow.

Yeah since my cells have processes which are easy to break I often end up having cellular debris and moreover induction of apoptosis causes more of these small grainy particles.

Thanks a lot.

Synchronize your cells??? I could not get the idea what it meant, thanks and hope i will get to know more about it later on. Thanks for your help. Sorry for replying late. Actually once I Saw the reply I am thinking to write again then I had some work and missed to reply. You have a nice time there.

Sanjaya

Hello Luisa,

I again started with flow cytometry and then recently got a result in which the viability of the cells is quite high, good for the control group but bad for the injury model......it could be that i lost my cells while centrifuging at 1000rpm in an attempt to decrease clumping of my cells for which I added EDTA to PBS and also decreased centrifuge speed greatly. I am not sure if if was the high confluency of the cells during treatment phase that the cells did not get injured much or the low centrifuge speed caused the apoptotic cells loss.

Provided that there are cell debris visible in the upper left quadrant would not the apoptotic cells have settled down.....the speed that settles cell debri should be enough to settle the apoptotic cells right......or also i could have lost much cell debri with apoptotic cells which i am not sure of.

http://img43.imageshack.us/img43/3915/20101220facs.jpg

Yeah another thing is I wanted you to comment on the plot that I observed- does the adjustment seem right, or needs some modification as of anything u can see in the graph. Thank you

@Nicole...mam could you please let me know when am analysing dna content in freshly harvested non fixed cells through PI..what does my graph indicates....i see 100 % population divided into G1, S and G2/M phases..since the acquiring is not done by myself having a little problem interpreting the data...since PI would only permeabilbize in the dead population how do i analyze the or extrapolate my results to DNA content analysis for live cells??

@luisa...mam how do we analyse viable cell without fixation on FACS using PI?