Flow cytometry Acquisition for HMVEC-L

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TJ
TJ's picture
Flow cytometry Acquisition for HMVEC-L

Hello everyone.  I have been trying to set acquisition paramaters for analysis of HMVEC-L.  I am using a BD FACSCalibur with FL1 and FL2  channels.  My problem is that most of my cells appear to be hitting the y-axis.  Anyone out there have suggestions for setting the SSC and FSC in addition to your experiences with FL1 and FL2 with compensation?  I would really appreciate any suggestions. 

Thanks,
TJ

Omai
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Hey TJ,

Hey TJ,

I routinely run flow on HUVECs. Endothelia are very large cells. I use E -1 for a FSC setting. If you play with the voltage a little you should be able to see your cell population on an X-axis FSC, Y-axis SSC dot plot. The SSC setting is around 500. They won't be mashed up against the y-axis.

For compensation advice, I need to know more about your 2 color fluorescence staining. Compensation is necessary when your FL-2 signal (usually PE tagged) is so bright it bleeds into your FL-1 window (FITC) that you get a false positive in your FL-1 signal. Can you tell me what flurophores you are using. Otherwise, to set for compensation, you need to have an unlabelled sample, a FL-1 (FITC) only labelled sample, and an FL-2 (PE) only labelled sample. This way you can use an FL1 vs FL-2 dot plot, set your negatives, and see if your single positive FL-2 stain is giving you a positive signal in the FL-1 window. If so, you can use compensation to diminsh that false positive signal. (FL2 _ %FL1)

Also, you could use an APC tag (runs in FL-4) to avoid all compensation issues.

Let me know some more specifics so I can help.

Omai

TJ
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Dear Omai,

Dear Omai,
Thank you for this info.  In terms of compensation- I am nucleofecting my cells with a plasmid containing a viral gene fused to GFP and am analyzing for apoptosis in these cells by using a substrate that is cleaved by active caspases.  The substrate fluoresces red.  I can't give you more information since Oncoimmunin (the company we purchase the substrate from) doesn't give much info on the fluorophore. 

marcus muench
marcus muench's picture
 You need to run all the

 You need to run all the possible controls to set up your machine.  Negative controls, unstained cells, will let you set the voltages so you can see all your cells on screen.  This is where you make sure they are not squished up on the y-axis.  If you brightest staining is not that bright feel free to bring up your negative control voltages so that in the end you are using as much of the real estate on your graphs as possible.  this gives you the maximum resolution.  Use single stained cells (transfected) for each of the colors to set compensation, so that the positive cells are only fluorescing in the correct channel.

TJ
TJ's picture
Thanks O Genic. 

Thanks O Genic.