Hello!
For a PBMCs culture I prepared a media with RPMI-1640, NaOH, B-ME, antibiotics and Fetal bovine serum. After extraction of the PBMCs from whole blood, I washed them twice with alsever then resuspended them in the media previously prepared.
While homogenizing, the media turns sticky/viscous, creating little strands.
I later tried pipetting it and put it in some alsever, but it won't mix.
If someone has an explanation, I'll be glad to know about it!
Thanks.
Hello Dounipher Douyon
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 commonly 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 cultured for 5-7 days 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.
What is the purpose of adding NaOH to RPMI media? That could be causing the media to turn viscous. Wash the cells preferably with sterile PBS.
Best.
In brief, most likely your cells are lyzed due to inappropriate conditions of pipetting, osmolarity or pH of solutions. You may preform visual inspection even under low magnification. Small round cells - OK, Clumps, stringy stuff, debris - something gone wrong.
P.S.: If your RPMI contains phenol red, what is the colour of media after adding all the components? If it turns from red into violet (or, less probably, yellow), it may be the pH issue.
Thank you for your answer !
And you're probably right because the media turned violet when I added NaOH in. I checked the ph, it's at 11.