Hello
I have performed LDH Assay on cell lines treated with various potentially toxic compounds at a range of dosage. I am trying to observe a time- dosage response. I allow my cells (BEAS-2B) to be treated with my compounds for 24 hours and 48 hours respectively in a 96 well plate assay. I am using pierce LDH Cytotoxicity Assay Kit (Thermofisher Scientific) to make the analysis. According to the manufacturer, I am supposed to make the calculation as follows:
Compound Treated LDH Activity- Spontaneous LDH Activity/ Maximum LDH Activity- Spontaneous LDH Activity X 100
where ,
Spontaneous LDH Activity= Activity of the Untreated cells (or 10uL of sterile water) &
Maximum LDH Activity= 10uL of 10X Lysis Buffer (provided by the manufacturer), allowed to incubate with triplicate of cells for 30min before proceeding with the rest of the assay
I also include the Positive Control (10uL of Triton-X 100) in triplicate for each of my experiments. I normally add this at the same time I am adding my compounds and hence allow to react with my cells at 24 hours and 48 hours respectively.
However, since I am trying to gain the LDH release value at 24 or 48 hours, the LDH shelf life, once released in the medium is said to be around 9 to 24 hours. Since I am planning my experiments at 24 hours and 48 hours, then there is a high possibility that LDH, once released into the media after treatment with my compound, would already be degraded before I make my readings. Hence, to normalize my calculations, what should I do?
Perhaps, I should try to make the calculations with my Positive Control (which I add at the same time as I add my compounds) instead of making the readings with the Maximum LDH Activity?
I tried to follow both calculations and found the values vary a bit in each case. When observed under the microscope, I noticed that all of my cells were killed when treated with Trition-X. This means I get to have maximum LDH release in that case as well.
What is the correct methodology to calculate my LDH release percentage in above mentioned case?
Thanks
You can use the formula below.
Hope this helps!
All the best!!
Cássio Prinholato da Silva Thanks for answering. My concern is what to keep as a maximum control over here? The control which u add at the same time as your drug/ chemical or the control which you add half an hour before adding the "reaction mixture"?
Hello, in my opinion the correct is to stick with Maximum LDH value to your calculation. Because you need as reference always a comparison with the maximum that your cells can release in the medium and not with time-dose. The time-dose effect you will observe for your other samples.
Bests,
Dear Faria Khan , the Maximum control is your positive control( Triton-X 100 in this case). Sometimes Triton can kill most cells (or all).
For this question: " (...)I tried to follow both calculations and found the values vary a bit in each case. When observed under the microscope, I noticed that all of my cells were killed when treated with Trition-X. This means I get to have maximum LDH release in that case as well?(...)"
If the cells had their membranes broken, yes!
Cássio Prinholato da Silva Thanks for the answer and explanation. I tried making the calculations based on Triton-X 100 treated cells as a Maximum Control and it worked for me!
- Badges
- Science topic
- Similar topics
- Biological Science
- Immunology