14 replies to this topic

[canotto]

Do somebody have a good protocol for CFSE staining ?
thanks

[SuMi]

I use the CellTrace CFSE kit from Molecular Probes and so this is the protocol that I use, although I have modified it for use with bacteria

http://probes.invitr...pis/mp34554.pdf

[canotto]

SuMi, on Jun 28 2009, 02:13 PM, said:

I use the CellTrace CFSE kit from Molecular Probes and so this is the protocol that I use, although I have modified it for use with bacteria

http://probes.invitr...pis/mp34554.pdf

could you please tell me where I can find a protocol for bacteria?
thanks

[illuminated]

I've got also a few questions about the CFSE staining. So far, I've found plenty of different protocols and some instructions seem to be even contradictory. So the questions are:

1) When putting CFSE to the cells, can I use normal DPBS or does it have to be PBS without Mg and Ca?

2) When suspending CFSE in PBS, does it have to containe BSA (or FCS) or will this even adversely affect the staining? Some protocols recommend the addition of 0.1% BSA to PBS, while other require completely protein-free solution as proteins would inhibit CFSE labeling. On one of the websites I found the explanation that for small cell numbers, CFSE is too toxic and so BSA has to be added in order to alleviate the toxic effect, but for large cell numbers this is not the case. What is your experience with it?

3) Do you incubate cells with CFSE at 37 C or at room temperature? And how long is the incubation time?

4) How many times do you wash the cells after labeling? And with what do you wash them?

5) Before flow cytometric analysis, do you fix the cells or not? If yes, do you use ethanol or paraformaldehyde fixation?

6) Do you use co-stainings? If yes, with which other dyes do you label the cells? Do you have a protocol for it?

[SuMi]

1. I always use PBS without Ca and Mg. I don't know if they adversely effect staining.

2. I don't use BSA for CFSE staining. My bacterial cells are resuspended in PBS (-Ca -Mg) and then I add the CFSE to the bacterial cell suspension. The CFSE itself is lyophilized and is reconstituted in DMSO. (The CFSE from Invitrogen is resuspended in 18µl DMSO to give a stock concentration of 5mM CFSE).

3. I incubate my bacteria with CFSE at room temperature in the dark. Usually for 20 mins. I I normally put my tube on a shaker at 300rpm during the staining time just to make sure the CFSE is evenly distributed.

4. I wash my cells twice after staining using PBS.

5. I don't fix my cells before flow.

6. I only do CFSE staining.

@ canotto:
For bacteria I resuspend my lyophilized CFSE in DMSO as outlined above to give a stock of 5mM concentration. I then add 2µl of this stock to 1ml bacterial suspension. The bacteria can be at any concentration. Mine are usually either 1e8/ml or 1e9/ml. Stain for 20-30mins at room temperature in the dark. Wash twice in PBS and resuspend bacteria in 1ml PBS.

[canotto]

SuMi, on Jul 24 2009, 04:59 PM, said:

1. I always use PBS without Ca and Mg. I don't know if they adversely effect staining.

2. I don't use BSA for CFSE staining. My bacterial cells are resuspended in PBS (-Ca -Mg) and then I add the CFSE to the bacterial cell suspension. The CFSE itself is lyophilized and is reconstituted in DMSO. (The CFSE from Invitrogen is resuspended in 18µl DMSO to give a stock concentration of 5mM CFSE).

3. I incubate my bacteria with CFSE at room temperature in the dark. Usually for 20 mins. I I normally put my tube on a shaker at 300rpm during the staining time just to make sure the CFSE is evenly distributed.

4. I wash my cells twice after staining using PBS.

5. I don't fix my cells before flow.

6. I only do CFSE staining.

@ canotto:
For bacteria I resuspend my lyophilized CFSE in DMSO as outlined above to give a stock of 5mM concentration. I then add 2µl of this stock to 1ml bacterial suspension. The bacteria can be at any concentration. Mine are usually either 1e8/ml or 1e9/ml. Stain for 20-30mins at room temperature in the dark. Wash twice in PBS and resuspend bacteria in 1ml PBS.

as for number 3 : do you incubate in PBS, PBS 0,1% FCS or medium?

[miBunny]

1.  Either PBS or DPBS should be fine.  We always used calcium and magnesium free DPBS but it really shouldn't matter.

2.  If BSA is present, it will "eat up" the CFSE (the BSA will be dyed green).  If you are staining a small number of cells, this will help prevent toxicity from over staining.  Cells are not particularly fond of CFSE staining and too intense staining will cause cell death.  If you are staining a large number of cells, the CFSE will just be soaking up the dye.

3.  I incubate lymphocytes for 15 minutes at 37 degrees.  

4.  I wash one to two times with complete media (10% FBS).  

5.  The cells do not need to be fixed before analysis.  If I can't analyze right away, I will fix with a 2-4% paraformaldehyde solution.  I have never tried another fixation method.

6.  The tricky part about costaining is getting the compensation right.  CFSE spills over big time into the other channels.  I have had my best luck with (this is on a FACs) dyes that are spectrally far apart from the green (APC or alexa 647 have never been a problem).  I knew one person that got a 4 color Facs stain to work with CFSE but the compensation and set up was a nightmare!

[canotto]

miBunny, on Aug 6 2009, 03:35 AM, said:

1.  Either PBS or DPBS should be fine.  We always used calcium and magnesium free DPBS but it really shouldn't matter.

2.  If BSA is present, it will "eat up" the CFSE (the BSA will be dyed green).  If you are staining a small number of cells, this will help prevent toxicity from over staining.  Cells are not particularly fond of CFSE staining and too intense staining will cause cell death.  If you are staining a large number of cells, the CFSE will just be soaking up the dye.

3.  I incubate lymphocytes for 15 minutes at 37 degrees.  

4.  I wash one to two times with complete media (10% FBS).  

5.  The cells do not need to be fixed before analysis.  If I can't analyze right away, I will fix with a 2-4% paraformaldehyde solution.  I have never tried another fixation method.

6.  The tricky part about costaining is getting the compensation right.  CFSE spills over big time into the other channels.  I have had my best luck with (this is on a FACs) dyes that are spectrally far apart from the green (APC or alexa 647 have never been a problem).  I knew one person that got a 4 color Facs stain to work with CFSE but the compensation and set up was a nightmare!

how do you stain adherent cells? do you use the same protocol ?

[miBunny]

I have not stained adherent cells.  I would probably start by trypsinizing the cells and staining them in suspension.

[canotto]

miBunny, on Aug 7 2009, 04:33 AM, said:

I have not stained adherent cells.  I would probably start by trypsinizing the cells and staining them in suspension.

thanks, but do you know if it is feasible and how to stain adherent cells? I'm asking because I was following this protocol http://probes.invitr...pis/mp34554.pdf but it didn't work on my PC-3 cells

[miBunny]

What kind of results did you get?

[canotto]

miBunny, on Aug 11 2009, 03:50 AM, said:

What kind of results did you get?

I think that I didn't stain the cells, because I couldn't find any fluorescence in FL1. What I tried it was to add 10 ml of 5 uM CFSE to a petri dish with PC-3 cells attached, leave it at 37 degrees and then replaced it with RPMI 10% FCS, leave 30 min and the replace with other RPMI 10% and leave for 3 days

[Astarte Biologics]

I think that I didn't stain the cells, because I couldn't find any fluorescence in FL1. What I tried it was to add 10 ml of 5 uM CFSE to a petri dish with PC-3 cells attached, leave it at 37 degrees and then replaced it with RPMI 10% FCS, leave 30 min and the replace with other RPMI 10% and leave for 3 days
[/quote]
That should work.  It is definitely feasible and the way you did it should have worked.  Were you comparing the labeled cells to unlabeled cells?  Cultured cells often have a lot of autofluorescence but with 5 uM CFSE you should see a big difference between stained and unstained.  If you collected the PC3 cells using trypsin it is also possible you trypsinized the label right off the cells.  You might want to try labeling in the dish like you did and in parallel label in suspension.  Don't wait 3 days, just go ahead and analyze using unlabeled cells for a baseline control.

Good luck.

[Binchen]

Hi!

I used CFSE before to stain T cells and DCs, but now I want to track Mycobacteria (BCG) with it for >14days in vivo.
The Mycobacteria have quite a thick cell wall and are sticky guys. I found a paper in which they label M. tuberculosis with CFSE and they state they use the normal PBS with 0.05% Tween 80 and stain for 60min at 37°C. What they don't say, unfortunately, is what concentration of CFSE they use. I guess you would need more than for DC or T cells because of the thick cell wall? And they also stain a lot longer than we do for DC. For DC we use 1uM CFSE for 10 min  at RT and then wash them twice.
Has anyone here tried staining mycobacteria with CFSE? Any suggestions for the concentration? I would like to be able to still see them after 2-3 weeks (and I don't know how fast the dye is fading in non-dividing cells) but I don't want to kill my BCG off with overloading them with CFSE. Unfortunately, trying different concentrations and looking at CFU will take ages as they take 3 weeks to grow to colonies on the plates...
Can anyone help me?

Thanks,
Binchen

[canotto]

do you have any plot of FSC/SSC and FSC/CFSE on ConA stimulated PBMCs ?

Since I don't have an anti-CD3 antibody, I don't understand exactly where to put my lymphocytes gate ( of course, stimulated lymphocytes are bigger than nonstimulated, but I don't understand how much. Do they overlay with macrophages?)

Thanks