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Question about FACS of maize protoplasts

FACS maize protoplasts

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#1 jerrywu1987

jerrywu1987

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Posted 20 February 2017 - 08:24 AM

Hi, I was wondering if anyone has experience on fluorescence-activated cell sorting (FACS) of plant protoplast. After PEG-transfection of the fluorescence probe into the maize protoplasts, I started to run the flow cytometry. 

 

The probe can produce green fluorescence once it binds to its specific mRNA target, and the fluorescence is clearly visible at nucleus of protoplast, and autofluorescence is not significant. 

 

However, when I sort the protoplasts based on green fluorescence, even the negative control gave me a strong fluorescence as showed in enclosed figure. Normally, the SSC-H (height of peak of individual event) and SSC-A (area of peak of individual event) should be proportional, that is, almost all the dots should fall in the diagonal. But the figure showed many off-target events. My explanation is that the protoplasts tend to form clumps, so that more than one protoplasts may drop together as one single event, so the SSC-A (area) value might be proportionally greater than corresponding SSC-H value, resulting in the dot off-targeting. 

 

It is really misleading when attempting to sort the positive events, because the probe treatment group also showed very similar pattern (see enclosed figure).

 

I filtered the protoplasts twice before submission to flow cytometry, but I don't know why there are still a lot of clumps. Maybe those off-target events were due to debris of dead protoplasts due to PEG treatment. I tried centrifuge to remove the the debris but apparently, the result was not that good. 

 

Do you have any suggestion to solve my issue, especially, how to reduce the clumps and dead cell debris in the living proroplast population, and get better separation of probe-positive events?

Thank you very much!

 

Attached Thumbnails

  • Negative Control.PNG
  • Probe treatment.PNG






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