Attached is picture of my results. Column 2-5 are drug treated cells.
Column 7,7 control
Col 8 is medium and presto blue only.
Starting from row 2-7
Is it normal that the values are too small? Also, the medium values higher than the column 2?
Here is my protocol:
1. I seed cells in 96 well plate 24 hr
2. Treat with drug
3. After 2 and 3 days, I do presto:
Mix 90 ul/ well medium plus 10 ul presto
Discard old medium and add presto medium mix making sure no bubbles
Incubate 30 -60 minutes
Use micro plate reader as follows:
-setting: two wave length 579,600
-lm1-Lm2
-reduced display.
Dear Heba,
It seems the assay has not worked at all. All wells seem displayed absorbance values close to that of the blank control (column 8). This means that the reagent did not undergo the expected reaction, as the result of which you should have obtained fluorascent signal. There may be several reasons for this;
1. Check the stability of the reagent. If the shelf life is expired you will need to purchase a new one.
2. Check the recommended storage temperature. If you have not stored the kit at proper conditions it may precipitate, which leads to insufficient distribution of the reagent to individual wells when you perform the assay. Check for crystals before use and ensure that the solution does not contain any precipitate at the time of its usage.
3. Although the manufacturer defines recommended values of Lm1 and Lm2, your sample may deen some optimization of the excitation and the emission wavelengths due to the differences in your cell culture medium. Check if your can perform spectrum scan in your multiplate reader device. You can perform fluorescence measurements with control cells at several Lm1. At each Lm1 do perform spectrum scan for the emission wavelength in the range defined by the manufacturer. This way you can establish the optimal Lm1 and Lm2 for your samples.
4. Also, check which protocol you perform with the instrument - fluorescence monochromator or fluorescence filter. You may need to adjust Lm1 and Lm2 accordingly.
5. Are your cells adherent or suspension-type? If your cells grow suspended in the medium, you may have discarded them together with the old medium.
6. Do you wash the wells with PBS before applying presto? Ensure that the PBS and cell culture medium during the assay are prewarmed to 37 degrees. Cold PBS may cause detachment and loss of cells. Also stress conditions may affect the metabolic activity of your cells.
7. Generally, kits like this need to be handled protected from direct exposure to light. Ensure that you don't expose your samples (or the reagent) to any bright light source. Shut down the light of the hood during sample preparation. Cover your sample with an aluminium foil during incubation.
8. Ensure that there is no contamination in your cell culture.
I hope you will find the answer of your question.
Thanks a lot Dr Mostafa this helped a lot. Can plz elaborate on number 3? Yes I want to change the reading. Also I think I do absorbace and I want to do flourescnce but how to know if my micro plate reader can do fluorescence and how to choose wave length.
Hi,
Just wanted to ask if you were able to solve the problem with your assay. If yes, could you please let me know what helped?
I'm having same troubles with my PrestoBlue assay
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