Ons for all situations are shown (n = 6 mice/condition). Bar = 200 mm.Ons for

Ons for all situations are shown (n = 6 mice/condition). Bar = 200 mm.
Ons for all conditions are shown (n = 6 mice/condition). Bar = 200 mm. (C) Measurement of white pulp location in hematoxylin/eosin-stained frozen spleen sections (3 sections/mouse, six mice/condition), quantified with ImageJ computer software. Mean 6 SD; Kolmogorov-Smirnov test, ***p,0.001. doi:ten.1371/journal.pone.0072960.gFlow cytometry analysis of immune cell populationsSecondary lymphoid organ cells from p110dWT/WT, p110dD910A/D910A, reconstituted p110dWT/WT and p110dD910A/D910A mice had been processed and stained for flow cytometry analysis (see Supplement S1).Flow cytometry evaluation of spleen stromal cellsStromal cells have been extracted making use of an established protocol [40]. Briefly, mouse CCR2 manufacturer spleens have been removed, pierced with fine forceps, and placed in Caspase 11 Biological Activity ice-cold RPMI-1640 (5 min, on ice). Spleens have been dissected, RPMI-1640 removed, and replaced with 2 ml of a fresh enzyme mix composed of dispase (0.8 mg/ml; Gibco) andPLOS 1 | plosone.orgp110d in Spleen Stromal CellsFigure 2. Absolute numbers of spleen and LN total cells, CD4+ and CD8+ T cells prior to and right after antigen stimulation. Spleens and LN had been extracted from p110dWT/WT, p110dD910A/D910A, and reconstituted mice in homeostatic circumstances (t = 0) and just after antigen stimulation (five days post-injection of inactivated C. albicans, t = 5 d). Entire organ cell suspensions had been counted to determine total cell quantity (A, D) and stained to establish CD4+ T (B, E) and CD8+ (C, F) cell numbers by flow cytometry (n = six mice/condition). Mean 6 SD. doi:ten.1371/journal.pone.0072960.gcollagenase IV (0.two mg/ml; Roche). Tubes had been incubated (37uC, 20 min), the cell suspension removed and placed in a fresh tube with ice-cold FACS buffer (3 FBS, 2 mM EDTA in PBS). The remaining spleen was re-incubated with 2 ml fresh enzyme mix (37uC, 10 min), just after which the cell suspension was removed and added to fresh tube above. The remaining spleen was reincubated (37uC, 15 min) in 2 ml fresh enzyme mix with vigorous pipetting every five min, the cell suspension was removed, placed in the same tube, whose contents were then filtered through a 100 mm nylon mesh. Cells were counted and viability assayed making use of trypan blue.Cells were stained with CD45 (30-F11, Biolegend), TER119 (TER119, eBioscience), gp38 (eight.1.1, eBioscience) and CD31 (MEC 13.three, BD Biosciences) in 100 ml (30 min, 4uC) before analysis on a Cytomix (Beckman Coulter).Stromal cell enrichment and cell sortingStromal cells were harvested as above. After spleens had been totally digested, cells were centrifuged, counted, and the single cell suspension depleted of non-hematopoietic stromal cells applying CD45 microbeads inside the autoMACS method (Miltenyi) andPLOS A single | plosone.orgp110d in Spleen Stromal CellsFigure three. Absolute numbers of spleen B cells and DC prior to and immediately after antigen stimulation. Spleens had been extracted from p110dWT/WT, p110dD910A/D910A, and reconstituted mice in homeostatic situations (t = 0) and following antigen stimulation (five days post-injection of inactivated C. albicans, t = 5 d). B cell (A) and DC (B) were stained and cell numbers determined by flow cytometry (n = 6 mice/condition). Mean 6 SD. doi:ten.1371/journal.pone.0072960.gp110dD910A/D910A mice and analyzed SLO in homeostatic circumstances. Lethally irradiated p110dWT/WT and p110dD910A/D910A mice have been reconstituted with total bone marrow from p110dWT/WT donors. Six weeks immediately after reconstitution, mice have been sacrificed for immunofluorescent staining of spleen and LN sections to detect immune cell populations (Figure 1); we.