How to get rid of contamination in human PBMC culture? - Been having problem in culturing human PBMC (May/21/2008 )
Hi. I have been trying to culture human PBMC isolated from whole blood. I haven't had any problems until the last month. I have been having contamination with fungus, bacteria, yeast and who knows what else. I have tried EVERYTHING i could think of to prevent the contamination. I work in the laminar hood, flame the bottle necks everytime i open and close, wear mask, swab everything with alcohol, make sure i UV the hood for 30 mins before i start my work. I even change and autoclave my pipette tips and filter my media and whatever reagents everytime i culture my cells. I use pen-strep, gentamycin and amphotericin in my media. I've done all the extra precautions that i can think of but i am still getting contamination in my PBMC culture. This has been going on for a month. The funny thing is, i dont get this problem with my cell lines. Please, can anyone help me with this? I really dont know what else i am doing wrong. 
Quite funny. May be the person whom U r getting the blood from has the infection
Quite funny. May be the person whom U r getting the blood from has the infection
A thing I can suggest is to use the complete PBMC media on some other cell line-- of course cells would not like it, but you will know if the media had a problem. If it has, then you can whittle it down to anywhich bloody component.
Don't put those cells in the general incubator, though .
. Quite funny. May be the person whom U r getting the blood from has the infection
A thing I can suggest is to use the complete PBMC media on some other cell line-- of course cells would not like it, but you will know if the media had a problem. If it has, then you can whittle it down to anywhich bloody component.
Don't put those cells in the general incubator, though .
.Thanks for the reply. i have used the same complete media on K562 cell line and it didnt give me any problems, no contamination.
Could it be from the laminar flow hood? i have heard that laminar flow hood can be a source of contamination. But i wiped the hood with 70% ethanol before working.
Sorry to hear that Ellyphant. Sounds like the blood is infected. Maybe you should change your source? But to have it infected with so many agents either means that the path lab is filthy or your donor is one sick puppy!
Sure sounds like a source contamination, I am guessing that the blood is handled in a non-sterile way before you get it. Yeast and bacterial contaminations in primary culture often come from skin.