I was trying to isolate fibroblastic reticular cells (FRC) and follicular dendritic cells from mice spleen, but kept getting poor yield. I found from literature that the frequency of FDC is around 0.2% in spleen but I could not find data about FRC. What is the expected frequency of FRC in mice spleen? Thanks.
You can injeact the spleen with warm phosphate buffer saline and incubate the spleen for 30 minutes at 37, then mix the spleen content well then withdraw the content.
Hello, Vincent!
Please You look at this article:
Origin and differentiation trajectories of fibroblastic reticular cells in the splenic white pulp
https://www.nature.com/articles/s41467-019-09728-3
Supplementary Figure 5. Identification of splenic fibroblastic stromal cell subsets
Fig. 5
As indicated in the reference supplied by Alexandr above, they are relatively low in number, depending on the precise region of the white pulp. I am not sure what you are using for an isolation protocol, but I would suggest not relying on the standard protocol for isolation of cells from spleen, as these cells will easily be lost using standard approach to the spleen. https://www.frontiersin.org/articles/10.3389/fcell.2019.00001/full
" Murine adult or 6 day old spleens were dissociated by pressing between microscope slides before filtering through a 70 μm strainer. The stromal fraction was collected in a tube containing 2 ml collagenase IV extraction buffer [(2% fetal calf serum, 1 mg/ml collagenase IV (Sigma-Aldrich) and 40 μg/ml DNase I (Sigma-Aldrich) in RPMI] and incubated for 20 min at 37°C with rotation. Two ml collagenase D extraction buffer [2% fetal calf serum, 1 mg/ml collagenase D (Roche Applied Science: North Ryde, NSW, Australia) and 40 μg/ml DNase I in RPMI] was added for another 20 min incubation at 37°C with rotation. An additional 2 ml of collagenase D extraction buffer was added and the cell suspension incubated for 20 min at 37°C with rotation. The activity of collagenase was halted by addition of 60 μL of 500 mM EDTA to give a final concentration of 5 mM. Digested spleens were washed twice with 5 ml sDMEM (300 g, 4°C, 5 min), and viable cell count determined using trypan blue staining. The cell pellet was resuspended in 1ml sDMEM for antibody staining, flow cytometric analysis and sorting. "
You can injeact the spleen with warm phosphate buffer saline and incubate the spleen for 30 minutes at 37, then mix the spleen content well then withdraw the content.