LDH-Cytotoxicity Assay Kit II (500 assays) (ab65393)

Can samples be frozen after the "Sample Incubation" steps?
I'm optimizing the treatment time point, but I'm not sure that I'll need to analyze all of my time points and I don't want to waste reagents. Will the LDH be stable after freezing and thawing?

ANSWER:

Thank you for your call yesterday and for your patience while I've been in touch with the lab.
I've heard back from one of the lab scientsts, who sent the following reply:
"The efficiency of the assay might be compromised if the supernatant from such an advanced step is frozen for later use. But I cannot say that for certain, since we haven’t tried using stored sups with this assay.
This is my recommendation for your experiment:

For optimizing the time point, you can have 1 sample for each time point initially and assay it all the way. That would prevent the waste of any excessive reagents and yet give the best results possible with this kit."
I hope that this information will be useful, but please let me know if you have any further questions and I'll be happy to help.

In regards to ab65393,please advise the sensitivity and the dymanic range (for some reason these are not indicated in the datasheet as for ab102526).
Thank you very much for your help.
Have a nice week,

ANSWER:

Thank you for contacting us. I apologize for the delay, while I was in contact with lab.
The ab65393 is a cytotoxicity assay. Therefore the sensitivity and dynamic range depends on the individual conditions and expt. Please use at-least 2 x 10^4 cells for each condition.
The ab102526 the sensitivity is 1 mU/ml and the dynamic range is 1-100 mU/ml.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
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Product code: 65393
Inquiry: For ab65393: please advise the sensitivity and the dymanic range. The customer would like to use the supernatant of the cells that grew in the suspension. The supernatant had already been collected and is stored at -80C. Will the kit be suitable for this kind of the starting materail? For ab102526: The customer would like to use the supernatant of the cells that grew in the suspension in the medium that contained 10% heat inactivated FCS. The supernatant had already been collected and is stored at -80C. Kindly advise if this will work? Thank you very much for the assistance. Best Regards,

ANSWER:

Thank you for contacting us.
Yes, the kit ab65393 will be OK to use with frozen samples. However fresh samples always provide best results. If in case the frozen sample gives erratic reading we would recommend trying the fresh samples also.
ab102526. Fetal calf serum does have LDH in it so it will interfere with the assay. We recommend using the culture medium with low %age (1%) of FCS or serum free medium for this assay. Alternatively you can use 10%FCS containing medium as control and then you can subtract the readings.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.
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Phone call requesting information on what buffer the LDH standard comes in.

ANSWER:

Thank you for contacting us and sorry for the delay in getting back to you.
The 20 uL of LDH provided with the kit is in a solution of 50% (NH4)2SO4. I hope this information has been of help.
If you require any further information, please do not hesitate to contact us again.

Hello, I intend to use this kit to quantify for LDH release into the culture media due to cell damage. I do not intend to perform cell lysis but check the effect of cell culture process on cell damage. For this I believe I can take the supernatant out of cell culture flask and incubate it with reagent and record the absorbance. But my concern is what should be the low and high controls for such experiment as I don not wish to take cells out of the flask. Thanks.

ANSWER:

Thank you for contacting us with your question, and for your patience while I have been in touch with the lab scientists regarding your enquiry.
According to the lab, the high control will need to be lysed cells in order to calculate the percentage of toxicity with the given equation. The protocol suggests plating cells at a constant density in wells of a 96-well plate in order to produce a controlled experiment, and you could simply remove media from the flask and run the reaction however there is not a recommended method to quantify this data. If you have access to cells that are not in your sample flask, I would recommend creating high and low controls as stated in the protocol, and plating these cells at approximately the same density as the cells in your flask to ensure consistent cell density.
I hope this information will be useful, but please let me know if you have any further questions and I will be happy to help.