Hey, I am currently doing my thesis and I am working with skin cells to detect a specific biomarker. I want to look for a biomarker in my collected media after treating them with 4 different treatments. However, my supervisor said that we would run them undiluted and diluted. So, how would I approach that ? Should I run a plate with undiluted samples and another plate where all samples are 1:2 diluted? So, for each sample should I add 100ul of the supernatant with 100ul of new fresh media to obtain this dilution and then run both plates to see if the concentration of my biomarker is detectable on the standard curve.
Please I need a full answer and some explanations.
Thank you
Hello Aya Al-rawashdeh
If you have enough wells, you may run both the undiluted as well as the diluted samples (1:2) in the same 96-well plate. I would suggest you also include one more dilution (1:4) in the run.
Run the standards as well as the samples (both undiluted and diluted) in triplicates. In case the wells are not sufficient, you may use another 96-well plate. Sample volumes are usually between 50-100μl; however, this can vary from assay to assay. I suggest you use 100ul of undiluted sample and for the diluted sample (1:2), add 50ul of the supernatant to 50ul of fresh media. Samples that generate values which are higher than the highest standard need to be repeated using a higher dilution factor.
Note that sample values should fall within the assay’s dynamic range. Values outside the range of the standard curve are usually non-linear, and because of this property it is not possible to extrapolate a value correctly using this curve.
You may also note that a standard curve should be run for every plate which means each plate should have a standard curve. If you cannot afford to run the standard curve for every plate because of limited supply or cost, at least you should run one standard curve with each set of plates for that day. If you are using serum-containing medium, it is necessary that you run an uncultured medium blank to obtain the baseline signals which can then be subtracted from the cultured media sample data.
Good Luck!
Hey Malcolm,
Thank you for your early respons. Your answer is quite beneficial and complete. Yes, I usually have one plate as a start just to see if I have any effetc, but in the near future I think I will have more samples which will require more wells. Hence, additional plates and I will be running a standard for each plate.
For my serial dilution, I abstract 450-570 from the blank or negative control standard. However, for the cultured samples, I run a cell media blank specifically for the type of cells I have.
I have 4 samples or treatments in triplicates and standards in duplicates. So, if three dilutions, undiluted, 1:2, and 1:4, I will need 36 wells and 48 wells for standards because 8 samples, each will be run in duplicate and three dilutions.
For the cell media, I will just have triplicates so three wells.
Should I dilute with vell media or blocking buffer??
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