Working with BioTek Synergy MX Monochromator-based multi-mode microplate reader, I found same dificulties, can anyone help me?
I would like to know what plates is normal to use to determine fluorescence (330,440/ 330,405). Or can I determine the type of plate I'm using?
What is the minimum volume recommended?
Thank so much
If you are doing a top read, then a black plate is typically used for fluorescence measurements. The exception to this is for time-resolved fluorescence, when it is preferable to use a white plate.
as above: black plate for fluorescence. But be carefull for fluorescence from the material of the plate itself if you excite in UV. Smaller cavities as in 384well-Plates are less usefull in my opinion, because of low signal on high background.
If you use less volume, you could focus to get highest intensity.
Black glas plate give low background, but they are really expensive, and they max not work properly with some protocols.
I tried using a SARSTEDT MICROTEST plate 96 well, F but it gave high values in wells that were empty! I can't relie in the rest of the values. Can I define the type of the plate in the software? the equipment is a BioTek Synergy MX Monochromator-based Multi-mode microplate reader. Thank so much for your aswers!
The plate you used is a transparent polystyrene plate. You should use a black plate. The reason is that the black material prevents plate fluorescence and absorbs scattered light.
You excite at 330nm and observe emission at 405nm/440nm?
For fluorescence you should use black plates. Using transparent plates you got also higher crosstalk. If you got wells with fluorescence close to wells without, you get signal even there. To get an idea, you could also measure an emission spectrum with the plate reader and compare it to you dye.
The measurement is 330/10nm excitation and 405/10nm emission. the problemis that I get high fluorescence in empty wells far from those wells with fluorescence.
Thank you so much for your feedback. I'm trying to find someone who uses black plates to try.
hi - did you compare signals from empty (i.e. dry) wells to those with just water or buffer (i.e. negative controls, background)? we see a strange phenomenon where adding buffer (water/HBSS) to wells actually reduces their fluorescence... in black plates and clear plates.
matt
I actually observed the same in my assay in which i am using a black well plate. The empty wells were showing higher flourescence than the wells that contain solutions
I´m now using black plates from eppendorf and I have good results.
Empty wells have almost zero fluorescence!
I recommend this black plates
Hi Carla,
the plates from eppendorf... were these plates made from polystyrene or from polypropylen?
Best Nick
So what i use are black costar plates to prevent auto-fluorescence and prevent false results. I don't know plate you are using. Normally the assay you are performing recommends a certain volume which is adjustable after the initial assay based on the results obtained
NIcholas, I have a related question. I am using the 96 black well plate but the problem is to define the correct probe height for the volume that I am using (100 uL). How can I understand which is the best for me? Thanks a lot
Carla, did you use Microplate 96/F, wells black, PCR clean, white, 80 plates (5 bags × 16 plates) Catalog No. 0030601700 ???
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