I am trying to enrich pDCs from mouse spleen. Following a bunch of papers I've collected from pubmed, I did an Optiprep gradient centrifugation followed with pDC enrichment kit from Miltenyi. The yield is usually low even considering the low numbers of pDCs in a naive mouse. Then I thought about a picture of pDCs. It looks exactly like an activated lymphocytes and hence the name "plasmacytoid". So they got to be medium or high density cells like lymphocytes, right? Am I losing all pDCs by targeting low density cells? I understand a conventional DCs should be low density considering its big volume.
Could someone please shed some light on this? Is there a good protocol on isolating pDCs from mouse spleen?
I don't have expirience with mousce ppDCs, but human are isolated on Ficoll (1.077 g/mL), while Optiprep is 1.006 g/mL. I don't think it will be isolated with low density fluid. Please find our cells counted in synovial fluid from children with JIA. We found so many pDCs (CD303+CD123+), so they are visible on FSC/SSC dotplot.
Hi,
from mouse spleen the more efficient way is to use the miltenyi kit with anti-mPDCA1-biotin+streptavidin beads. You can use total splenocytes directly for the purification. The optiprep gradient is not good since you loose and damage a lot of cells.
Best
Julien DIANA
I would suggest reading
Cd11c+B220+Gr-1+ Cells in Mouse Lymph Nodes and Spleen Display Characteristics of Plasmacytoid Dendritic Cells
J Exp Med. 2001 October 15; 194(8): 1171–1178.
and
The Enigmatic Plasmacytoid T Cells Develop into Dendritic Cells with Interleukin (IL)-3 and CD40-Ligand
J Exp Med. 1997 March 17; 185(6): 1101–1112.
hope is helpful
ferdinand
Hi Dahui You,
I use optiprep gradient to enricher pDC with a final density of 1.068, containing 5 mM EDTA. I obtain the cells by centrifugation at 1700g for 10 minutes.
Usually I pass through all of this headache and FACS sorting the CD11c+ B220+ population. Very good viability and quality.
Normally I would sort my pDCs from the spleen. But being short on time I thought I would use the Miltenyi kit to obtain untouched pDCs (negative selection). I physically disrupt the spleen using scissors, a 70um mesh filter and a plunger from a syringe. However, my yield was terrible: From one Black 6 WT mouse spleen I got 7,000 cells! When I sort I get about 1-2x10^6/spleen. I have used the pDCA-1 beads as mentioned and those work much better. Also, using Streptavidin beads instead of the anti-biotin beads has worked better for me in the past as well.
Sorry- I mean that 7,000 was the worse yield I've ever had from miltenyi beads using the negative selection cocktail and anti-biotin beads. In the past I've used either: 1. negative selection with a biotin cocktail followed by Strepavidin beads or 2. PDCA1 beads for a positive selection. These two methods gave me better results with about 150K to 300K pDCs. This yield, though still much lower than what I get from sorting is the best I've been able to get using beads from one mouse spleen (C57/B6)