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

Ons for all conditions are shown (n = six mice/condition). Bar = 200 mm.
Ons for all circumstances are shown (n = 6 mice/condition). Bar = 200 mm. (C) Measurement of white pulp location in hematoxylin/eosin-stained frozen spleen sections (three sections/mouse, six mice/condition), quantified with ImageJ software. Imply six SD; Kolmogorov-Smirnov test, ***p,0.001. doi:ten.1371/journal.pone.0072960.gFlow cytometry evaluation of immune cell populationsSecondary lymphoid organ cells from p110dWT/WT, p110dD910A/D910A, reconstituted p110dWT/WT and p110dD910A/D910A mice were processed and stained for flow cytometry analysis (see Supplement S1).Flow cytometry evaluation of spleen stromal cellsStromal cells were extracted making use of an established protocol [40]. Briefly, mouse spleens were removed, pierced with fine forceps, and placed in 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.eight mg/ml; Gibco) andPLOS One particular | plosone.orgp110d in Spleen Stromal CellsFigure two. Absolute ERK Compound numbers of spleen and LN total cells, CD4+ and CD8+ T cells before and just after antigen stimulation. Spleens and LN had been extracted from p110dWT/WT, p110dD910A/D910A, and reconstituted mice in homeostatic conditions (t = 0) and following antigen stimulation (five days post-injection of inactivated C. albicans, t = 5 d). Entire organ cell suspensions have been counted to identify total cell number (A, D) and stained to decide CD4+ T (B, E) and CD8+ (C, F) cell numbers by flow cytometry (n = six mice/condition). Imply six SD. doi:10.1371/journal.pone.0072960.gcollagenase IV (0.2 mg/ml; Roche). Tubes were incubated (37uC, 20 min), the cell suspension removed and placed in a fresh tube with ice-cold FACS buffer (three FBS, two mM EDTA in PBS). The remaining spleen was re-incubated with two ml fresh enzyme mix (37uC, 10 min), 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 5 min, the cell suspension was removed, placed inside the very same tube, whose contents have been then filtered via a one hundred mm nylon mesh. Cells have been counted and viability assayed applying trypan blue.Cells were stained with CD45 (30-F11, Biolegend), TER119 (TER119, eBioscience), gp38 (8.1.1, eBioscience) and CD31 (MEC 13.three, BD Biosciences) in 100 ml (30 min, 4uC) ahead of evaluation on a Cytomix (Beckman Coulter).Stromal cell enrichment and cell sortingStromal cells were harvested as above. Following spleens have been totally digested, cells were centrifuged, counted, and the single cell suspension depleted of non-hematopoietic stromal cells utilizing CD45 microbeads within the autoMACS system (Miltenyi) andPLOS One | plosone.orgp110d in Spleen Stromal CellsFigure three. Absolute numbers of spleen B cells and DC just before and after antigen stimulation. Spleens had been extracted from p110dWT/WT, p110dD910A/D910A, and reconstituted mice in homeostatic situations (t = 0) and after antigen stimulation (5 days post-injection of inactivated C. albicans, t = 5 d). B cell (A) and DC (B) have been stained and cell numbers determined by flow cytometry (n = six mice/condition). Mean 6 SD. doi:10.1371/journal.pone.0072960.gp110dD910A/D910A mice and analyzed SLO in homeostatic situations. Lethally D3 Receptor Synonyms irradiated p110dWT/WT and p110dD910A/D910A mice had been reconstituted with total bone marrow from p110dWT/WT donors. Six weeks following reconstitution, mice have been sacrificed for immunofluorescent staining of spleen and LN sections to detect immune cell populations (Figure 1); we.