Why are my cells dying in MLR?

I am isolating MDSCs from frozen healthy PBMCs, I am using MACs to get a population that is CD14+ and HLA-DR-.  After isolation, I culture the isolated MDSCs with Autologous PBMCs in RPMI 1640  with Pen/Strep solution, 2 mM L-Glutamine, 50 uM 2-Mercaptoethanol and 10% heat inactivated FBS. I noticed they start to loose their CD14 expression approx. 5hrs after isolation. Any help in how to maintain CD14+ and HLA-DR- MDSCs after isolation would be appreciated.

Thanks!

Hey, all!

So, I am trying to expand NK cells from PBMC. It seems simple enough, but I am wondering what the ratio of PBMC: irradiated K562-41BBL-IL-15 should be. I did 0.5x10^6 PBMC: 1.5x10^6 K562 cells, and I am culturing them in medium containing 200 U per ml IL-2. Will this work well? 

Thanks, folks!

A.

I am going to measure protein expression of BCL-6, BLIMP-1, IRF-4 and XBP-1 in PBMC or B cell of healthy controls by flow cytometry, but i don't know protein level of these molecule in control...

Hello all,

I am going to treat primary blood eosinophil with PAF.

However, when I searched for that product from ENZO, I saw different PFA products with different molecular structures, e.g. carbamyl-PAF, 1-O-Hexadecyl-2-O-acetyl-sn-glycero-3-phosphorylcholine, 1-O-Palmitoyl-2-O-acetyl-sn-glycero-3-phosphorylcholine, ect...

Could you please suggest me what kind of PFA suitable for treating primary blood granulocytes, especially eosinophils?

Thank you very much for any help from you all.

I am working on stimulation of PBMCs isolated from HIV positive individuals using 10%FBS RPMI and HIV gag p24 (1µg/ml) for three days to investigate some inhibitory receptors (Like PD-1) immunocenescence (CD57) and immunoactivation (CD28) markers on the surface of T cells by flow cytometry. I have seeded 300,000 cells in each well of 24-well cell culture plate and I have not harvested more than 1 million cells pulling up five wells after 3 days. I would like to know if three days is not too much to culture and then how can I generally increase the number of cells.

We have stimulated our PBMCs from normal controls with PMA (5-50 ng/ml) and Ionomycin/ 1ug/ml. Our target is to assess the functions of NK cells (both cytokine release and cytotoxicity) in these donors. No matter what we decreases the conc. of PMA or Ionomycin we observed that our NK cells dies. I have been using PMA/Ionomycin for long time to stimulate T cells and gamma delta T cells and we did not encounter any problems !

What is the alternative could we use to stimulate NK cells ?

Hello all. 

B cells isolated from chick embryos' bursa do not survive in culture for very long. (down to 30% viability after 9 hours of incubation). I really wanted to test the effect of demethylating the DNA on several aspects of that cell. Problem is, 5-azac works on "trapping" the enzymes in charge of methylating the DNA (DNMT), so the cells have to go through at least several cycles for that material to take a proper effect. Protocols I have seen use 5-azac on cultures of cell lines for a minimum of 24 hours, and most of them use it for 3 days. This is not an option for bursal B cells. Does anyone have any experience with faster acting demethylators? 

Thank you

I am culturing murine PBMCs with CFSE for 4 days along with ConA (1ug/20uL) and I have an unstimulated control but when I read my cells on flow the CFSE signal from my unstimulated cells is around where my stimulated cells are.  I've been trying to figure out what I am doing wrong and thought maybe it might be my culture media or something, but I use DMEM +10%FBS, 1% P/S and L-glutamine.  I isolate my cells through RBC lysis, could that be causing them to become stimulated?  I'm not certain where else to start looking so any tips would be greatly apprecaited.

Hi Everyone,

I am culturing the monocytes derived from the PBMC of the patients blood samples. After two weeks of the culture growth, some of the cells appeared as shaded (or in background looks like covered by a layer of something else), [pic 1- day 1, pic 2- day 6, pic 3- day 13 and pic 4- day 21] imaging at 0.4x.

Is the cells need passaging? If yes, I am worried weather the these cells will be adhered again or not. But as per the protocol, no need to do the subculture.