Ons for all circumstances 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 region in hematoxylin/eosin-stained frozen spleen sections (3 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 CCKBR Formulation analysis (see Supplement S1).Flow cytometry evaluation of spleen stromal cellsStromal cells have been extracted applying 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 had been dissected, RPMI-1640 removed, and replaced with two ml of a fresh enzyme mix composed of dispase (0.8 mg/ml; Gibco) andPLOS A single | plosone.orgp110d in Spleen Stromal CellsFigure two. Absolute numbers of spleen and LN total cells, CD4+ and CD8+ T cells ahead of and immediately after antigen stimulation. Spleens and LN were extracted from p110dWT/WT, p110dD910A/D910A, and reconstituted mice in homeostatic circumstances (t = 0) and right after antigen stimulation (five days post-injection of inactivated C. albicans, t = five d). Whole organ cell suspensions were counted to figure out total cell quantity (A, D) and stained to determine CD4+ T (B, E) and CD8+ (C, F) cell numbers by flow cytometry (n = 6 mice/condition). Mean 6 SD. doi:10.1371/journal.pone.0072960.gcollagenase IV (0.2 mg/ml; Roche). Tubes have been 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 2 ml fresh enzyme mix (37uC, 10 min), immediately 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 each five min, the cell suspension was removed, placed inside the exact same tube, whose contents had been then filtered via a 100 mm nylon mesh. Cells had been counted and CCR9 medchemexpress viability assayed applying trypan blue.Cells have been stained with CD45 (30-F11, Biolegend), TER119 (TER119, eBioscience), gp38 (8.1.1, eBioscience) and CD31 (MEC 13.3, BD Biosciences) in 100 ml (30 min, 4uC) before analysis on a Cytomix (Beckman Coulter).Stromal cell enrichment and cell sortingStromal cells have been harvested as above. Following spleens had been totally digested, cells have been centrifuged, counted, along with the single cell suspension depleted of non-hematopoietic stromal cells using CD45 microbeads within the autoMACS technique (Miltenyi) andPLOS One particular | plosone.orgp110d in Spleen Stromal CellsFigure three. Absolute numbers of spleen B cells and DC ahead of and following antigen stimulation. Spleens had been extracted from p110dWT/WT, p110dD910A/D910A, and reconstituted mice in homeostatic conditions (t = 0) and immediately after antigen stimulation (5 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 = six mice/condition). Imply six SD. doi:ten.1371/journal.pone.0072960.gp110dD910A/D910A mice and analyzed SLO in homeostatic conditions. Lethally irradiated p110dWT/WT and p110dD910A/D910A mice were reconstituted with total bone marrow from p110dWT/WT donors. Six weeks just after reconstitution, mice had been sacrificed for immunofluorescent staining of spleen and LN sections to detect immune cell populations (Figure 1); we.