Troubleshooting forum on flow cytometry http://www.protocol-online.org/forums/index.php Fri, 20 Nov 2009 03:51:58 -0800 3600 http://www.protocol-online.org/forums/index.php?showtopic=11678 Does anyone have an experience of a 3-colour labeling for flow cytometer with only one excitation argon-ion 488 nm laser? I need to stain DNA and two antigens by indirect immunofluorescence (or, in other experiments, DNA, 1 antigen and CFDA-SE). There is no problem with FL1 (CY2, AlexaFluor488, DyLight 488-conjugated antibody, or CFDA-SE), in my case they need minimal or no compensation in FL2. But it appeared difficult to use simultaneously a red DNA-binding stain and a red antibody. PI is not good at all, as it gives fluorescence in FL2, FL3 and FL4. I tried to use PE-conjugated antibody (FL2) and 7-aminoactinomycin D (7-AAD) for DNA (in FL4). However, in reality 7-AAD yelded in low resolution when used in 2 mkg/ml, and even in this relatively low concentration gave some FL2 fluorescence. Moreovere, it was hard to compensate for it since the concentration of 7-AAD was unsaturating and the positions of 7-AAD peaks varied from sample to sample. If anyone worked with 7-AAD, what concentration have you used? Do you feel it is a good pair indeed?
On the other hand, longer-wavelength stain LDS 751 (Molecular Probes) exists, but I am not sure if it is good for quantitative staining and if it can help to overcome these problems (we are thinking of buying it if it is really better than 7AAD).
As an alternative I thought about exchanging the chanels: to buy PerCP-conjugated secondary antibody (Jackson ImmunoResearch) to view them in FL4, and to use some DNA stain for FL2, but I do not know any good FL2 DNA stain with no fluorescence in FL4 (do you know?)
Do you know anything about these or other alternative pairs?
Thank you!]]> Thu, 19 Nov 2009 11:41:48 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11678 http://www.protocol-online.org/forums/index.php?showtopic=11573 Do you have any plots/histograms?
]]> Mon, 16 Nov 2009 06:40:22 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11573 http://www.protocol-online.org/forums/index.php?showtopic=11523 its been a frustating weeek for me, I have a cell population which are nearly 70% positive for GFP, upon fixing using the BD cytofix, and there by washing with BDcytoperm, I lose the GFP content to 30% or less, makeing me difficult to stain with some antibody hence hindering the multiplex reaction, Really dont know how to tackle this issue, one way it quench the GFP completely but how?, or are there suitable ways to fix without detoriating the GFP content! please guide with your useful suggestions. Thanks in advance.]]> Fri, 13 Nov 2009 11:28:09 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11523 http://www.protocol-online.org/forums/index.php?showtopic=11494 I would like to know if there is a free software (equivalent to CellQuest) for performing cell cycle analysis of FACS files aquired by FACScalibur
thanks]]> Thu, 12 Nov 2009 11:10:17 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11494 http://www.protocol-online.org/forums/index.php?showtopic=11469
I'm going to do several FACS experiments using between 2 and 4 antibodies. My question may appear maive but I'm wondering if I should add each antibody to each sample separately or if I can make an antibody mix and then add the mix to each sample. Is there a risk that the antibodies bind to each other in the mix or not?
I also read a topic about not using trypsin to detach cell in culture when using Ab against membrane receptors? My targets are not receptors but are protein membrane so I guess I shouldn't use trypsin neither?
Thanks in advance for any advice ]]> Wed, 11 Nov 2009 09:17:57 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11469 http://www.protocol-online.org/forums/index.php?showtopic=11447
I have been working with HaCaT cells and now I am measuring apoptosis with these cell lines.

I am using vybrant apoptosis kit method to analyse apoptosis in these cell lines.

I have some questions regarding this....

1. I am getting lot of debris during my analysis

2. I am also finding a lot of early apoptotic cells


I will be glad if you could answer me these questions..

]]> Tue, 10 Nov 2009 10:13:16 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11447 http://www.protocol-online.org/forums/index.php?showtopic=11323 does any one have suggestion how to detect dapi with flow cytometry? should it be one plot (FL4) or two plot (SSC/ FL4)?
can flow cytometry detect the apoptotic bodies using dapi staining?
many thanks
]]> Tue, 03 Nov 2009 09:16:49 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11323 http://www.protocol-online.org/forums/index.php?showtopic=11300
I wanna stain GFP transfected human cells [adhaerent monolayer culture] with DAPI or Hoechst. My problem is that every fixation kills my GFP in the cells. Does anyone know a good protocol for vital staining of the nucleus without any fixation of the cell.


Thx Neo]]> Mon, 02 Nov 2009 05:31:26 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11300 http://www.protocol-online.org/forums/index.php?showtopic=11266
I had strange results, I couldn't compensate between FDA ( read in FL1) and PI ( read in FL3). Do you have any plot as example of this analysis?

]]> Fri, 30 Oct 2009 13:44:06 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11266 http://www.protocol-online.org/forums/index.php?showtopic=11260 I would like to know if isotype controls have to be used
at the same concentrations of specific antibodies.

Thanks in advance!]]> Fri, 30 Oct 2009 11:02:27 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11260 http://www.protocol-online.org/forums/index.php?showtopic=11210
In haste as I was about to finish a long day of experiments (19hr day of continous work) by running some splenocytes on a cytometer, I forgot my box of antibodies on the table at room temperature for 3 hrs. The box is opaque so they are not exposed to light.

I use colors such as

FITC
APC
APC-Cy7
PE
PE-Cy7
DAPI
pacific blue

Most of these antibodes are brand new. I came back after a 3h of flow session and realized my mistake and put them back immediately at 4C in the fridge. Although, now I am freaked out if the antibodies have degraded.

Can someone tell me if all hope is lost ? WIll those conjugated dyes degrade within hours at room temp? It was a maximum of 3h. I remember reading an old publication that most normal flourochromes such as FITC are stable at room temp for like 700h or so before the loss of signal.

I am dying with dread if those Ab have degraded. I worked hard to standardize that panel and my supervisor won't be very happy to hear if they degraded due to such a stupid error.

thanks,

V]]> Wed, 28 Oct 2009 23:50:01 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11210 http://www.protocol-online.org/forums/index.php?showtopic=11201 I have a killer toxin, which generates pores on susceptible yeast cells, so I want to make a viability test with PI. Now, I incubate the sensitive cells with the toxin for 12 and 24 hour, but the problem is that I can't afford to make the measurments with the flow cytometer at each sampling time. The question is that, after the sampling and staining, can the cells be stored at 4ºC simply, or I have to fix them with 70% ethanol or formaldehyde (which one do you prefer?). Will not the PI diffused from the dead cells at 4ºC overnight? I suppose it won't. What do you think?
Thanks in advance.]]> Wed, 28 Oct 2009 16:09:59 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11201 http://www.protocol-online.org/forums/index.php?showtopic=11156
i am new in learning flow cytometry however I tried to detect the expression of a membrane protein that i transfected to generate stable line selected using hygromycin and detected the expression of the clones in FITC labeled antibody.

I detected the expression of two batches of the clones and the expression level of my target protein in these stable clones (most of the population) reached 85-97% . However, after two days, my labmate verified my findings after expanding the cells in 10 cm dish, and detected it in flow again and the expression level of our target protein did not match my findings! ALmost all clones does not have any expression of the target protein.

I do not understand this finding, what could be the possible reasons for this? did i set up the wrong parameters? are my control cells the problem? is my staining the problem? i tried to follow diligently all the protcol, i just do not understand what could be the problem. I am bothered because I got good results at first and second run and turned out to be wrong in other person's hand who has been working with flow for many many years. I wanted to know possible reasons for this because I dont want the credibility of my result to be questioned.

I hope you understand, i wanted to learn and wanted to know what might have caused this unreproducible result.

thank you sooo muchh....]]> Tue, 27 Oct 2009 08:28:46 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11156 http://www.protocol-online.org/forums/index.php?showtopic=11154 Here's the scenario. Let's say I have 200k cells per ml in wells. Sample A is the control and Sample B is treated with a known substance that kills 90% of the cells at 48 hours. When I run flow at 48 hours, do I: use the same volume as the control to read or do I count out the same number of cells between the control and treated samples and read those samples (which would be 10x of the treated sample)? Readings would be done on PI or 7aad and several antibodies for expression.

I have a few thoughts but I keep on going back and forth on this and am unsure. If I'm not too clear, let me know - it's something I'm working on Thanks.]]> Tue, 27 Oct 2009 08:04:38 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11154 http://www.protocol-online.org/forums/index.php?showtopic=11035
I am working with a cell line which is terribly clumpy. It is maintained on fibronectin-coated dishes and there are two types of populations: floating clumps and adherent ones. Both types are equally sticky/clumpy after digestion with trypsin-EDTA (Invitrogen's). In fact, I could see the single cells moving towards each other and forming doublet/triplets and more, within 10 mins of post-flushing with medium+FCS.

Any suggestions as to how I can successfully maintain a single cell suspension long enough for sorting?

Thanks!]]> Wed, 21 Oct 2009 23:46:48 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11035 http://www.protocol-online.org/forums/index.php?showtopic=10963
I have seen used Camptothecin or H2O2 for apoptosis, and Triton x100 or heat ( 44 degrees) for 90 min for necrosis.

Do you have any suggestion?]]> Mon, 19 Oct 2009 12:30:17 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10963 http://www.protocol-online.org/forums/index.php?showtopic=10933 I have to titrate some fluorochrome-conjugated antibodies for flow cytometry experiments and I'd like to solve some doubts.
If I titrate an antibody with a cell concentration of 2*10^5 cell/100ul, do I need to titrate it again when using a cell
concentration of 5*10^5-10^6 cell/100ul?

Thanks a lot!]]> Sun, 18 Oct 2009 05:00:57 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10933 http://www.protocol-online.org/forums/index.php?showtopic=10931 I've read that adding DNase I during or after PMA/Iono stimulation can avoid the formation of clumps, thus improving flow cytometry analysis. Do you have experience with this protocol? Could you explain this to me in more details?

Thanks a lot!]]> Sun, 18 Oct 2009 04:41:41 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10931 http://www.protocol-online.org/forums/index.php?showtopic=10887
I'm going to do GUS staining to see the promoter activity of a gene. The solution I need should be 1mM X-Gluc+50mM Phosphate buffer+10% Methanol. I doubt my calculations because I SUCK in math.

Can anybody here do this calculation for me if I need 500uL of above mentioned staining solution from stocks of 500mM X-Gluc, 50mM phosphate buffer and absolute Methanol?

Pardon me for getting into a PhD without basic math skills !

Thanks]]> Thu, 15 Oct 2009 06:09:05 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10887 http://www.protocol-online.org/forums/index.php?showtopic=10840
Recently a doubt was raised in how to analyze facs data.

In this specific assay we stain cells for a specific antibody and of course also the isotype to correct for it.

We treat cells with different compounds and then analyze the percentage of positive cells and the mean/median of expression.

For that we set the quadrants in the isotype and then apply the same quadrant for the corresponding condition. the percentage of positive cells in a certain condition is subtracted by the percentage of positive cells of the isotype. Until here no problems.

The question is then how to compare different conditions...

for example if we have:

control
control isotype
compound 1
compound 1 isotype
compound 2
compound 2 isotype

Do you set the quadrants to the control isotype and apply the same to all conditions, or should i set independent quadrants for each (coumpound + iso combination)

the question was raised because treatment with these compounds changed significantly the percentage of positive cells in the isotype.

hope i was clear in the question


looking forward to hear from you

Cheers

hugo]]> Tue, 13 Oct 2009 16:16:03 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10840