The chamber slide was then placed into the temperature control chamber on the Leica SP5 microscope. Introduction encode members of the inhibitory class of heterotrimeric G-proteins so named based on their ability to inhibit adenylyl cyclase activity . Targeted loss-of-function mutations of have been generated in mice revealing redundancy as well as tissue specific functions for is flanked by loxP sites (recombinase. We crossed these mice to knock-in (KI) mice , thereby deleting a portion of the coding sequence in B cells and causing a loss of Gi2 in those cells. To determine the functional importance of Gi3 in B lymphocytes lacking Gi2 we crossed the mice to the Gnai3-/- mice. We compared B lymphocytes from (referred to as DKO) mice. This analysis provides insights into the importance of Gi2 and Gi3 for B cell responses to chemoattractants and B cell function. Materials and Methods Animals C57BL/6, and B6.SJL-Ptprca Pepcb/BoyJ mice were obtained from Jackson Laboratory. mice were kindly provided by Dr. Michael Reth (University of Freiburg, Germany). For bone marrow reconstitution, seven weeks old B6.SJL-Ptprca Pepcb/BoyJ (CD45.1) mice were irradiated twice with 550 rads for total of 1100 rads and received bone marrow from C57BL/6 CD45.2 mice (control) or from DKO C57BL/6 CD45.2 mice. The engraftment was monitored by sampling the blood 28 days later. The mice were used 6-8 weeks after reconstitution. All mice were used in this study were 6-14 weeks of age. Mice Tenofovir maleate were housed under specific-pathogen-free conditions. All the animal experiments and protocols used in the study were approved by the NIAID Animal Care and Use Committee (ACUC) at the National Institutes of Health. Cells Splenic B cells were isolated by negative depletion Tenofovir maleate using biotinylated antibodies to CD4, CD8, Gr-1 (Ly-6C and Ly 6G), and CD11c and Dynabeads M-280 Streptavidin (Invitrogen) as previously described . The B cell purity was greater than 95%. When needed B cells were cultured in RPMI 1640 containing 10% fetal calf serum Tenofovir maleate (FCS, Gibco), 2 mM L-glutamine, antibiotics (100 IU/mL penicillin and 100 g/mL streptomycin), 1 mM sodium pyruvate, and 50 M 2-mercaptoethanol. Cell culture media for S1P chemotaxis was same as above except charcoal-dextran filtered FCS was used. Flow cytometry, antibodies, and cell proliferation Single cells were re-suspended in PBS, 2% FBS, and stained with fluorochrome-conjugated or biotinylated antibodies against B220 (RA3-6B2), IgD (11-26c-2a), IgM (R6-60.2), CD24 (M1/69), Tenofovir maleate CD3 (145-2C11), CD4 (GK1.5), CD5 (53-7.3), CD8 (53-6.7), CD11c (HL3), CD11b (M1/70), CD138 (281-2), CD19 (1D3), CD38 (90), IgG1 (X56), CD93 (AA4.1), BP-1 (6C3), GL-7 (GL-7, Ly-77), CD95 (Jo2), CD21 (4E3), CD23 (B3B4), CD43 (S7), CD184 (CXCR4, 2B11), CXCR5 (2G8), CCR7 (4B12), CD11a (M17/4), CD29 (HMb1-1), CD49d (9C10, MFR4.B), CD54 (YN1/1.74), CD62L (MEL-16), 47 (DATK32), CD279 (PD-1, RMP1-30), CD45.1 (A20), or CD45.2 (104) (all from eBioscience, Biolegend, or BD Pharmingen). Biotin-labeled antibodies were visualized with fluorochrome-conjugated streptavidin (eBioscience). LIVE/DEAD? Fixable Aqua Dead Cell Stain Kit (Molecular Probes) was used in all Tenofovir maleate experiments to exclude dead cells. Data acquisition was done on FACSCanto II (BD) flow cytometer and analyzed with FlowJo software (Treestar). The cell proliferation studies were performed using the eFluor? 450 (eBioscience) in PDGFRB a standard dye dilution assay. Purified B cells were stimulated for 96 hours with various combinations of the following reagents: 1 g/ml CD40 (HM40-3), 1 g/ml LPS (Sigma-Aldrich), recombinant mouse IL-4 (10 ng/ml, R&D Systems), or 10 g/ml AffiniPure F(ab’)2 fragment goat anti-mouse IgM (Jackson ImmunoResearch Laboratories). Data acquisition was done on FACSCanto II flow cytometer. The percent of cell divisions were calculated using FlowJo software, which is defined as the proliferation indexes divided by the division indexes, and multiplying the results by 100, assuming no cell death. Chemotaxis assays Chemotaxis assays were performed using a transwell chamber (Costar) as previously described . Splenic B cells were immunostained for B cell subsets with fluorochrome-conjugated antibodies against B220, CD21, CD23, CD24, CD93, IgM and IgD, washed twice, re-suspended.