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• 11

  Ryan Mitchell

  09 June 2009 | 20:51

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  Must you lyse the cells to get a reliable reading? Can you infer anything from the read that you get from the adherent intact cells? Should I still have 100uL of fluid resting on the cells if I want to read GFP without lysing, for the purpose of keeping the cells alive?

  Thanks!

• 12

  Makoah Nigel Aminake

  10 June 2009 | 10:24

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  Hi Ryan, if you really need to keep your cells intact and alive, you can’t use a fluorometer reader, that is the problem…..you probably need another reporter, like Luciferase or B-galactosidase. measuring GFP in intact cells is not really possible, you wouldn’t find a significant difference between your wells.
  I hope I help in some way, feel free to post again

• 13

  saamarthy karunakar

  01 August 2009 | 19:40

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  Hi…

  I am doing a cell based assay using HaCaT cells (Human embryonic kidney cell line). I would like to know the ways that we can quantify the GFP expressed in each well of the 96 well plate.

  I have been using this Microtiter plate (Tecan M200) reader to measure GFP in live cells, but unable to gett the difference between the negative control and transfected samples.

  I am very much sure that all my samples express GFP in the cells.

  Could someone please help me out to and suggest a way to measure GFP in the microplate reader?

• 14

  Makoah Nigel Aminake

  02 August 2009 | 06:35

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  Hi saamarthy karunakar, measure GFP fluorescence in live cells is very difficult, i am not sure if someone has done that before. I always lyse cells, you may consider lysing the cells and measure using the fluorometer.

  Nigel

• 15

  Paula Nunes

  02 August 2009 | 21:17

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  Hi Karunakar,

  From the protocol you sent me you gave the step
  10. Read absorbance at 450nm- multiplate reader.
  If this is really what your settings were, then that might be the problem –
  GFP reading must be done in the fluorescence top read mode (depending on the machine this may be called different things, but absorbance methods will not work with GFP – you need fluorescence). If your machine has a bottom read mode is also not good the detector then reads through the plastic of the plate and the plastic of normal tissue-culture 96 well plates has a lot of background.

  If your machine uses filter sets you need a filter for the excitation around 488 nm and a filter for the emission around 535 nm for good spectral separation. Always check the bandwidth of your filter-sets too! I saw a posting above that mentioned the use of a 510 nm filter – depending on the bandwidth of the filter sets used you may even risk overlap with the excitation filter (Think about a 30 nm bandwidth around 490 nm = 485-505 nm – if you had the same around 510 it would be 495 – 525 nm).
  The following website has excitation/emission spectra and is a great resource for learning about fluorescence:
  http://www.microscopyu.com/tutorials/flash/spectralprofiles/index.html

  As for the person who asked about whether lysing is necessary, and what I found was that lysing gives you a more accurate difference – because the live cell is stationary and you usually only read the center of the well if your seeding is even or your transfection was more efficient in the center your reads will show less difference. For the case of non-adherent cells, you could try it with a shake before the read for example to see if that helps even things out. A caveat with lysing is your lysis buffer which a) may interfere with your fluorophore signal by quenching, denaturing etc, and b) it may be fluoresce or absorb at your desired wavelength. For this I recommend trying different lysis buffers with a positive control: a dilution series of a concentrated lysate made by mechanical disruption of cells you know are fluorescent in water for example, or loose dye like FITC, etc. See if you can decrease the concentration by 10 fold, if you also get a 10 fold decrease in your background-subtracted value. Using trypsin is ok – but again check your background! those pink solutions absorb for sure, and depending on the brand/your wavelength may even fluoresce. When quantifying, the background value of the solution must be subtracted.

  Anyway hope this helps!

  Paula

• 16

  saamarthy karunakar

  04 August 2009 | 07:39

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  Thank you Paula and Nigel.

  I will try what you have mentioned and let you know the outcome.

  karunakar

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