Hi, I seed 4.5 x 10^6 cells per well in 3ml complete RPMI 10% (1.5 x10^6 cells per ml) with 200ng/ml FLT3-L (in 6-well plates). 8 days later (no media change), I get about 10 million cells per plate, 20% of which are pDCs, as stained like Naik et al, JI, 2005. Hope this helps.

Hi, your protocol should work as you described it. An important factor is the type of FCS used. Depending on the batch there are huge differences in the outcome (from high percentage of pDC with high IFN-alpha producing capacity to no pDCs or no IFN-alpha production). Definitely worth trying a couple of different FCS batches. Harvesting the cells shouldn't be a critical step because you are just taking the suspension cells, centrifuge and they are ready for stimulation/analysis. 

For a detailed protocol refer to:  

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SourceAvailable from: Shalin Naik

 Article: CD8+, CD8-, and plasmacytoid dendritic cell generation in vitro using flt3 ligand.

Shalin H Naik, Meredith O'Keeffe, Anna Proietto, Hubertus Hochrein, Ken Shortman, Li Wu

Same as Asolina, I normally get equivalent amount of cells as input after an 8-day Flt3L BM culture. 

A few things that I follow (ref: Weimershaus M and van Endert P.  Methods in Mol Biol. vol960, p351-357):

• seed cells at 10^6/ml with 200 ng/ml Flt3L
• no RBC lysis (except for cell counting)
• use plastic petri dishes (same as in agar plates for bacterial culture, not treated for cell culture)
• add low conc of GM-CSF (300 pg/ml); increase to 1 ng/ml for the last 2 days.
• after harvesting floating cells, wash dishes with cold PBS twice and pool cells.

Hope this help.

Hello Michael, 

I am also facing a similar problem with my BM derived pDC. I have done with 100ng/ml of the ligand for 10^ cells/ml. And  cultured it  for 7 days. I then sorted them for a pDC specific marker. The frustrating fact is that initially I got a differentiation of about 65%. But now I am not getting the same rate of differentiation...now it is about 7-10%.

I was wondering as to how to proceed. And have inputs from some people in this area as to change the media and add fresh ligand every couple of days for a week. Will have to try out this protocol. Keeping my fingers crossed that this should work out for me ....the ligand is so costly that it scares me to play with that ....

And may I know on  what basis you harvest your pDC?  by FACS?

Hi everyone,

Thanks for your speedy replies.

I will take on what everyone has suggested, thanks very much!

The issue I have is during harvesting of the cells. As I said, I typically only recover ~10% of the cells I seeded (~1 x 106/ml). I don't observe a high proportion of dead cells, neither do I notice many cells remaining in the dish (only adherent cells) after harvesting the non- and semi-adherent cells.

During analysis of the cells (by flow cytometry) with antibodies against specific pDC markers, a very low proportion of the cells harvested are pDCs (again, ~10% of the 10% I harvested, if you understand me). This is another major issue, and I believe (as with many of your suggestions) that the culture plates and/or FCS could be to blame. I have now ordered other plates and will experiment with different FCS stocks. Once I know the outcome, I will be sure to update you all.

Thanks again for all your help.

Michael

Michael, have you checked whether your antibodies are working fine ?

Hi Michael!

I am also facing similar problem. Is it working for you now with modifications?

For Flt3L culture, would you prefer mouse or human Flt3L? I see that different labs use them indistinctly.