Ntation authorized all animal studies (refs 12021, 12022), in compliance with national and European Union legislation. All efforts had been created to lessen suffering.Bone marrow reconstitution assaysp110dWT/WT and p110dD910A/D910A mice had been lethally cirradiated (single dose, ten Gy). Soon after three h, mice have been reconstituted by intravenous injection (tail vein) of total bone marrow from p110dWT/WT mice. Six weeks after reconstitution, mice were sacrificed, and spleen and LN collected. Half were frozen for immunofluorescence research, along with the remainder used to prepare single-cell suspensions for populations counts and flow cytometry analysis.Immune response induction with heat-inactivated Candida albicansHeat-inactivated Candida albicans cells (106) have been injected into p110dWT/WT, p110dD910A/D910A, reconstituted p110dWT/WT and p110dD910A/D910A mice (see Supplement S1 for specifics). Mice were sacrificed five days post-injection, and spleen and LN collected. Half had been frozen for immunofluorescence research, along with the remainder used to prepare single-cell suspensions for populations counts and flow cytometry analysis (see Supplement S1).Immunofluorescence of SLO sectionsFrozen sections of spleen and LN from p110dWT/WT, p110dD910A/D910A, reconstituted p110dWT/WT and reconstituted p110dD910A/D910A mice have been analyzed by immunofluorescence staining to study distribution and location of immune cell (Thy1.2+ and CD3+ T cells, MOMA+ MMM, B220+ B cells, CD11c+ DC, see Supplement S1).Hematoxylin-eosin staining of spleen sectionsFrozen spleen sections from p110dWT/WT, p110dD910A/D910A, reconstituted p110dWT/WT and reconstituted p110dD910A/D910A mice had been hematoxylin/eosin stained to analyze lymphoid follicle area (see Supplement S1).p110d in Spleen Stromal CellsFigure 1. Immunofluorescence analysis of immune cell distribution and white pulp area. Frozen sections of spleen and LN from p110dWT/WT, p110dD910A/D910A, and reconstituted mice had been immunofluorescence-stained to detect T cells (CD3+, Thy1.2+), B cells (B220+), MMM (MOMA+) and DC (CD11c+). Representative pictures of spleen (A) and LN (B) sections for all situations are shown (n = six mice/condition).NNZ 2591 Biological Activity Bar = 200 mm.Collagenase IV, Clostridium histolytica Biological Activity (C) Measurement of white pulp region in hematoxylin/eosin-stained frozen spleen sections (three sections/mouse, 6 mice/condition), quantified with ImageJ software program.PMID:24455443 Imply 6 SD; Kolmogorov-Smirnov test, ***p,0.001. doi:10.1371/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 evaluation (see Supplement S1).Flow cytometry analysis of spleen stromal cellsStromal cells were extracted working with an established protocol [40]. Briefly, mouse spleens had been removed, pierced with fine forceps, and placed in ice-cold RPMI-1640 (five 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 One particular | www.plosone.orgp110d in Spleen Stromal CellsFigure two. Absolute numbers of spleen and LN total cells, CD4+ and CD8+ T cells prior to and after antigen stimulation. Spleens and LN were 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 = five d). Whole organ cell suspensions have been counted to identify total cell quantity (A, D) and stained to establish.
