I collected blood from a dairy cow and used a typical PBMC isolation protocol. Briefly, I diluted the sample 1:1 with PBS and centrifuged, removed the buffy coat and added it to 30ml PBS and then overlaid Histopaque-1077 with the PBS+buffy coat, followed by centrifugation. I removed the buffy coat again and did a lysis and restore (sodium phosphate solutions) step to remove remaining red blood cells. I centrifuged the sample one more time and then dumped off the supernatant and added cRPMI (RPMI, antibiotic-antimycotic, heat inactivated FBS at 10%) and plated them at 5x10^4, 1x10^5, and 3x10^5 cells/ml in 1ml/well in a 24 well plate.
I did not stimulate the cells, just purely seeded them and let them sit overnight. When I went to extract the supernatant at 24h the cells seeded at 3x10^5 cells/ml were in a gelatinous type configuration. I don't understand. Any insight would be helpful. Cell culture contamination from the regular culprits has been ruled out-- no fungi, yeast, bacteria.
This has now happened to me on two different occasions with two different dairy cows from the same herd. The last time and this time I did a PBMC isolation, after the Lysis:Restore step the tubes were still pretty turbid after the centrifugation step (1500rpm for 10min) so I did this step twice (the second spin i bumped the speed to 1700rpm) and afterwards the liquid wasn't exactly clear, but I went ahead with my protocol. I don't know if that [the turbidity] matters, but thought it was worthy to mention. I just assumed it would be a loss of cells...
Could this be buffer related? Cow-dependent? Am I missing something?
Hope someone can help.
Jordan,
This gelatinization is probably the result of cell lysis and release of DNA. Your description indicates that it only occurs at the highest cell density, not at lower densities. Is that correct? If so, I can't explain that unless the turbidity is actually platelets and higher cell density is introducing a large enough number of platelets to trigger cell lysis. If you have not already done so, you should look carefully, using phase contrast microscopy or using a simple leukocyte stain, at your final cell suspension to ascertain what cell types are present. If platelets or PMN are present, these will certainly activate PBM.
You also indicated that you are not stimulating the cells during the 24 hour incubation. However you are using heat inactivated FBS in the media. hiFBS contains a large variety of leukocyte activators including huge quantities of fibronectin and significant amounts of PDGF, EGF, etc. If the culture period is only 24 hours, then you probably don't need FBS unless your experiment requires cell division to occur. To avoid these activators in FBS you could use purified (type V) endotoxin free bovine serum albumin at 1 or 2% instead.
Good luck!
Thank you! After doing several lyse and restore methods, I concluded that i was lysing the cells too long and then they ended up releasing DNA to cause the gel type substance.
I haven't thought about the FBS as an activator, but that provides me with further questions to my experiment in progress. Thank you for you insight!
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