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• 1

  Paula Nunes

  03 April 2009 | 15:02

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  Hi,
  I have used lysis/plate reader fluorescence for means of quantification
  of GFP in 96 well format in LLC-PK1 cells (another kidney epithelial
  cell line) and found it worked quite well. I am wondering what problems
  you have encountered with your lysis techniques? I can tell you about
  some issues we had and what we did to solve them:

  1) Cells sometimes came off the bottom of the plate during washing. If
  our cells are overconfluent they can detach more easily, so controling
  for confluency and pressing the multipipettor tip to the top of the well
  to reduce the pressure of the liquid hitting the cells seemed to help. I
  know HEK cells can detach very easily if too overconfluent. We had to
  monitor our cells by microscopy after every treatment step during the
  pilot studies to minimize this.

  2) The kind of lysis buffer needs to have low background otherwise you
  will underestimate number of cells in the well. I found RIPA had a
  higher background than the buffer I use now, and other simpler buffers
  that didn’t completelly lyse the cells. The one I use is a triton-based
  custom recipe I’d be happy to send to you if you are interested. Also
  improving the multipipettor technique can decrease well-well variability
  in volume – you can try using different tips for example. More flexible
  tips that make a tighter seal with your multipipettor decrease the
  variability in volume, which helps too.

  3) If seeding/washing is changing the number of cells per well
  dramatically, you could try normalizing by the DNA content of the cells.
  We use a 1:15,000 dilution of TOPRO3 in the lysis buffer itself, a
  far-red DNA dye that increases its fluorescence 200 fold when bound to
  DNA. This way, you read the well in two wavelengths, and it gives you a
  better idea of what is going on.

  Anyway hope that helps!

• 2

  Srisathiyanarayanan Dharmaiah

  04 April 2009 | 02:29

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  Hi Paula,

  Thanks alot for your time and reply. I used passive lysis buffer from Promega which we generally used to lyse HEK293T cells for doing a western blot. I don’t know if that would interfere!

  Yes, you are certainly true that cells come off and looks weired after the PBS wash. Since am using DMEM which has a phenol red indicator, I included this wash step before I go take my reading.

  I will use some of your suggestions and see if that works.

  If you can email me your working protocol, I appreciate. Thank again for your time and reply,

  Cheers

• 3

  Paula Nunes

  05 April 2009 | 12:23

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  Hi Sathiya

  So here are a couple things to try:
  1) You could try to grow the cells in phenol-free DMEM (both Invitrogen and Cell-gro offer these) – if you can just aspirate and avoid the wash step that may help.

  2) Try putting your vacuum on a partial setting to decrease the suction, which may be more gentle on the cells.

  3) You could also try putting your phenol-free DMEM in a column of an empty 96 well plate, aspirate it gently as you would for cells, and add your lysis buffer(+/-TOPRO3 if you want to try that), then read this on the plate reader – you can then subtract the mean of the fluorescence values and later subtract this background reading from your other readings. This will enhance the differences you see

  4) The lysis buffer from Promega may very well be great, just give it read on its own and see if subtracting that value helps, and that really being gentle on the cells and controlling for confluency (if that is possible for your assay) may just solve the problem. We optimized our protocol by seeding cells at different confluency (ie 5, 10 15 20 uL of cells seeded) and seeing which method gave us good differences in cell numbers. If you would like to try the lysis buffer I use, the reference is
  Hasler et al. J Am Soc Nephrol 16: 1571-1582, 2005. The buffer can be kept as a stock in the fridge without the NaF, Na3Vo4, triton and the protease inhibitors (which you can either substitute for a protease inhibitor cocktail or, if you are reading within 10 min of lysis, without the protease inhibitors) and those ingredients I add the day of the lysis step. The TOPRO3 I use at a 1:15,000 dilution.

  4) Finally if the cells coming off the plate is really a big problem, you could also try poly-lysine or collagen-coated plates to enhance cell attachment. (I know more expensive…but you can always ask BD or Corning for samples to be sure before switching)

  Anyway good luck to you!

  Paula

• 4

  Sudipta Tripathi

  16 April 2009 | 18:02

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  Hi
  You can do live cell GFP expression in FACS. I do it to determine viral titer based on GFP expression in 293T cells.
  The original paper is from Methods in Molecular Biology, vol. 183: Green Fluorescent Protein: Applications and Protocols by Nicoletta Eliopoulos and Jacques Galipeau
  If you can’t access the article let me know. I can send you an e-copy.

  Sudipta

• 5

  Srisathiyanarayanan Dharmaiah

  17 April 2009 | 04:49

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  Hi Sudipta,

  Thanks for your suggestion. I will look into the paper. Today I did the live cell imaging. I got a good, perfect and neat image by which we can say the expression level(induction or inhibition). But it was tough to quantify. I need to see the plate reader methods Paula suggested here. Will post further when I get through some interesting results. Please send me an ecopy to srisathiyanarayanan@gmail.com. I appreciate it.

  Sathiya

• 6

  Makoah Nigel Aminake

  25 April 2009 | 09:50

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  Hi Guys , I am happy to join this forum. Please Paula and Dharmaiah, could you send me your detailed protocol of how to quantify EGFP expressed in HeLa cells? I have been working on this for a month and I didn´t properly quantify the fluorescence with the fluorometer.My problem really on the lysis and handling the cells before measurement.

  Thanks for helping, my e- mail is : makoahnigel@yahoo.fr

• 7

  Srisathiyanarayanan Dharmaiah

  27 April 2009 | 21:49

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  Hi Makoah Nigel Aminake,

  I tried using the plate reader somehow I cannot really measure it. But I can see that its differential expression using BD pathway 855 live cell imaging. May be Paula can explain it better. When I tried using the plate reader I dont see any difference in the values between the wells GFP untransfected and transfected. I used Synergy2 (from Biotek) and Spectramax. I used 488 exitation wavelength and 510 emission wavelength. I don’t know how can I deal with the plate reader.

• 8

  Makoah Nigel Aminake

  28 April 2009 | 04:35

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  Thanks for the reply, I am really concern with the lysis step by step method,working always on ice do help, or combining triton 0.1% lysis with Freeze-Thaw or sonication. I am confuse. Probably GFP also is not expressed enough to be detected by the reader.

  I am waiting.
  Thanks again.

• 9

  Amit Kumar Singh

  28 April 2009 | 07:54

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  Sathiya,
  Do you know any thing about BRET and FRET assay?
  You can use Luminometer which have 96 well microplate reader.
  Cheers,
  Amit

• 10

  Makoah Nigel Aminake

  03 May 2009 | 10:24

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  hi guys, i finally figure out how to use the plate reader to measure GFP fluorescence, has to do with the way you lyse your cells, I use the triton in HEPES and was able to measure.

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