Hi researchers!
I want to run a mtt assay on PBMC to show that a cytotoxic drug in a leukemia, does not have any effect on normal cells I mean PBMC!
Is there any special protocol to isolate ,seed and treat Them?
does any one know how many cells can be seed in 96 well plate?
how about the drug concentration?IC 50 or less than IC50?which one is better?!
please help me,thank you
Hello !
-To isolate PBMC from total blood you need a gradient centrifugation over Ficoll (1,077) , you can check YouTube for a full protocol.
- in my lab we usually put 10⁶ cell/ml and we start a cell culture only if the cell viability is >90%
- for cell viability and enumeration i usually use Trypan blue.
- The drug concentration changes according to the drug itself , so if you are using a new drug for the very first time you need to do a cytotoxicity/therapeutic effect tests using different doses of your drug.
- You should put your drug solution in your culture well with the cells ( i usually add 20ul of my drug solution) , you can keep them for 24h in 37°c 5% CO2 or for 48h ...etc depends on what are you looking for.
Good luck ...
Hello Elmira Zarei
1. PBMCs are isolated by density gradient centrifugation, as different components of the blood have different densities and can be separated accordingly. The density gradient medium most used is Ficoll-Paque.
To isolate PBMCs, whole blood, diluted with PBS, is gently layered over an equal volume of Ficoll in a Falcon tube, and centrifuged for 30-40 minutes at 400-500 g without brake. Four layers will form, each containing different cell types—the uppermost layer will contain plasma, which can be removed by pipetting. The second layer will contain PBMCs and is a characteristically white and cloudy “blanket.” These cells can be gently removed using a Pasteur pipette and added to warm RPMI medium or sterile PBS to wash off any remaining platelets. The pelleted cells can then be counted, and the percentage viability estimated using trypan blue staining.
PBMCs can be in culture in 24- or 96-well plates, using RPMI-1640 medium supplemented with 10% FBS and 1% antibiotic solution, and incubated at 37°C in a humidified, 5% CO2 atmosphere. PBMCs do not readily proliferate without stimulus. So, to induce proliferation, phytohemagglutinin (PHA) or lipopolysaccharide (LPS) may be added to the culture medium at a concentration of 1-5 µg/mL.
2. When performing MTT assay, you may seed 1.0 x 10^5 – 5.0 x 10^5 cells per well in 96-well plate. Treat the cells with the drug candidate for 24- 48 hrs. depending upon the potency of the drug.
3. Use the drug concentration (IC50).
Best Wishes.
If your PBMC still have a reddish color you can wash with MilliQ for approx. 30sec to remove red blood cells
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