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Help me explain the FACS results


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#1 Jia

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Posted 06 November 2010 - 11:26 PM

Recently I run on a FACS analysis using simply the FITC conjugated antibody directly stained against one of the membrane proteins of my cells. I did twice. The first time I used almost 1:150 FITC conjugated antibody and the results showed no signals. So I stained my cells with diluted antibody at the concentration of 1:5, 1: 10 and 1:20 at the second time. Although the results showed very high positive signals, I doubted about it. It looked like the percentage of the positive cells decreased with the antibody concentration. The only explanation is that the antibody concentration is too high and most of the signals come from the background. Am I right?
2. I use the direct staining method and there is no way to provide controls such as cells which do not express the marker or staining my cells with the secondary antibody only, especially when you using the same graph for cell sorting.
Could anyone help me analysis the results and give me suggestions?
Thank you so much!
I attach the results also.
Attach 1: First time- 1:150 FITC Attached File  FITC.pdf   11.08KB   297 downloads
Attach 2: First tim-controlAttached File  control.pdf   10.74KB   216 downloads
Attach 3: Second time- Control Attached File  Global Sheet1_03112010142140.pdf   10.58KB   211 downloads
Attach 4: Second time-1: 5 FITC Attached File  Global Sheet1_03112010142201.pdf   11.07KB   216 downloads
Attach 5: Second time-1: 10 FITC Attached File  Global Sheet1_03112010142208.pdf   11.25KB   209 downloads
Attach 6: Second time-1: 20 FITC Attached File  Global Sheet1_03112010142214.pdf   11.35KB   200 downloads

#2 Rsm

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Posted 08 November 2010 - 01:50 AM

You are using primary cells, right? It seems that you have lots of debris, I would exclude all events below FSC 50,000. You can see two populations in your plot, your cells are most likely in your right population.
You have very obviously two populations of FITC positive cells, at least at the 1:10 and 1:20 condition. One is rather low positive, but clearly different from the other. I can't understand why you can not get a negative control, which is essential in your case. Why can't you use a cell line or splenocytes? You can not make a statement with this data that you show. You need a negative control, to say "this population is negative, and this one positive!". Your 1:5 dilution shows background staining. Do you block Fc receptors? Also, performing all staining reactions on ice reduces background quite well.

rsm
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#3 Jia

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Posted 08 November 2010 - 09:35 AM

Thank you so much for your reply!
1. Actually I use differentiated embryonic stem (ES) cells, therefore there are various cell types in it.
2. I agree with your opinion about the cell population. I see two populations also. Next time I am going to try different gates.
3. I am not sure if the population would be the same when I use a cell line that can not be stained with Mab. Even if I use a cell line as a negative control, still it is hard to say which population is the right one.
4. I am sorting my cells of interest. I do not know if I can use the blocking buffer to block the Fc receptor.








Recently I run on a FACS analysis using simply the FITC conjugated antibody directly stained against one of the membrane proteins of my cells. I did twice. The first time I used almost 1:150 FITC conjugated antibody and the results showed no signals. So I stained my cells with diluted antibody at the concentration of 1:5, 1: 10 and 1:20 at the second time. Although the results showed very high positive signals, I doubted about it. It looked like the percentage of the positive cells decreased with the antibody concentration. The only explanation is that the antibody concentration is too high and most of the signals come from the background. Am I right?
2. I use the direct staining method and there is no way to provide controls such as cells which do not express the marker or staining my cells with the secondary antibody only, especially when you using the same graph for cell sorting.
Could anyone help me analysis the results and give me suggestions?
Thank you so much!
I attach the results also.
Attach 1: First time- 1:150 FITC Attached File  FITC.pdf   11.08KB   297 downloads
Attach 2: First tim-controlAttached File  control.pdf   10.74KB   216 downloads
Attach 3: Second time- Control Attached File  Global Sheet1_03112010142140.pdf   10.58KB   211 downloads
Attach 4: Second time-1: 5 FITC Attached File  Global Sheet1_03112010142201.pdf   11.07KB   216 downloads
Attach 5: Second time-1: 10 FITC Attached File  Global Sheet1_03112010142208.pdf   11.25KB   209 downloads
Attach 6: Second time-1: 20 FITC Attached File  Global Sheet1_03112010142214.pdf   11.35KB   200 downloads



#4 Rsm

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Posted 08 November 2010 - 11:42 PM

Do you use MEF as feeder layer? Are they positive for your gene? You could try to differentiate them from ES cells (usually they would be CD34+) then you'd have an internal negative control. If you have a negative population, you can set your gate excluding all negative events, and thus defining positive events. A positive control would be good as well, can you overexpress your gene of interest? At least you could find out if your Mab is working well, or what concentration you need.
Blocking the Fc receptor is not necessary for cell cultures...
Do you stain on ice? it really improves signal/noise.

rsm
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#5 Jia

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Posted 10 November 2010 - 08:53 PM

Thank you so much!
That is a brilliant idea. Yes I use MEF as my feeder layer which do not express the genes of my interest. It should be a great internal control. Thank you so much again! I did put the sample on ice during the staining process. I will show updated files after using the modified methods.
Jia









Do you use MEF as feeder layer? Are they positive for your gene? You could try to differentiate them from ES cells (usually they would be CD34+) then you'd have an internal negative control. If you have a negative population, you can set your gate excluding all negative events, and thus defining positive events. A positive control would be good as well, can you overexpress your gene of interest? At least you could find out if your Mab is working well, or what concentration you need.
Blocking the Fc receptor is not necessary for cell cultures...
Do you stain on ice? it really improves signal/noise.

rsm



#6 almost a doctor

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Posted 11 November 2010 - 03:50 AM

Hi, what about isotype controls?

I'm assuming from your data that your control cells are unstained. Have you tried staining your cells with an IgG-FITC of the same isotype as the MAb you are using for staining your membrane proteins? That should help differentiate between specific stain and background.

I agree with the populations in the FSC-SSC plot. You should gate both populations and look for FITC expression on each of them.

Anyway, I think using isotype controls is essential for proper FACS analysis in order to show positive stain.

Good luck.




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