As such, potential actions of these bacterial metabolites on immune cells in the lamina propria cannot be discounted

As such, potential actions of these bacterial metabolites on immune cells in the lamina propria cannot be discounted. In the present study, we focused on the role of the high-affinity transporter Slc5a8 on the biology of dendritic cells (DCs). of age. Germ-free and age-matched conventional mice were obtained from Taconic Biosciences, Inc. (Hudson, NY, USA) and used as described previously [23]. Institutional Animal Care and Use Committee (IACUC) of the Georgia Regents University approved all animal procedures reported in this study. Isolation of DCs and their culture Mature DCs were isolated from mouse spleen using CD11c microbeads (Miltenyi Biotech, Auburn, CA, USA) followed by magnetic separation. Purity of isolated DCs was determined by flow cytometric analysis using CD11c antibody. DC purity for typical DC isolation was ~90%. DCs were then cultured in complete culture medium (RPMI 1640 medium, containing 10% fetal calf serum, 10 mM HEPES pH 7.4, 2 mM glutamine, 100 IU/ml penicillin, 100 g/ml streptomycin, and 50 M 2-mercaptoethanol) for 48 h. Isolation and culture of CD4+ T cells CD4+ CD25? CD44+ CD62LHi T cells were isolated from spleen and mesenteric lymph nodes of OT-II transgenic mice by FACS using fluorochrome-conjugated antibodies (both from eBioscience, San Diego, CA, USA). For in vitro FoxP3+ CD4+ Treg conversion assay, DCs pretreated with or without butyrate were recovered from culture at the end of 48 h and co-cultured for 4 days with these OT-II T cells at a ratio 1:2 in complete medium. The culture medium was supplemented with 0.5 g/ml Ovalbumin peptide (ISQVHAAHAEINEA), 0.4 ng/ml TGF and 5 ng/ml IL-2. The cells were then fixed with FoxP3/Transcription Factor Fixation/Permeabilization kit (eBioscience, San Diego, CA, USA) and stained with antibodies against CD4 and FoxP3 for analysis on LSR II flow cytometer. For in vitro IFN-+ CD4+ T cell suppression assay, DCs pretreated with or without butyrate were recovered from culture at the end of 48 h and co-cultured for 4 days with the CD4+ CD25?CD44? CD62LHi T cells from OT-II transgenic mice at DC:T cell ratio 1:2 in the complete medium. The culture medium was supplemented with 0.5 g/ml Ovalbumin peptide (ISQVHAAHAEINEA), 10 ng/ml IL-12, 10 g/ml anti-IL4, and 5 ng/ml IL-2. On day 4 of co-culture, cells were recovered and cultured further in presence of 5 ng/ml IL-2 for 48 h, after which the cells were stimulated with phorbol myristate acetate plus ionomycin in the presence of GolgiStop and Golgiplug for 5 h. Finally, cells were fixed with FoxP3/Transcription Factor Fixation/Permeabilization kit (eBioscience, San Diego, CA, USA), stained with antibodies against CD4, CD25, and IFN- and analyzed on LSR II flow cytometer. In vivo IFN-+ CD4+ T cell suppression assay OVA-specific CD4+ CD25?CD44? CD62LHiCD90.1+ (Thy1.1) T cells from OT-II donor mice were injected (i.v.) into recipient WT or (Thy1.2) mice 1 day before immunization. Mice were then immunized with a mixture of ovalbumin dissolved in PBS and complete Freuds adjuvant at 1:1 ratio (s.c.). Two weeks later, animals were sacrificed to obtain cells from spleen. These cells were then stimulated with phorbol myristate acetate plus ionomycin in the presence of GolgiStop and Golgiplug for 5 h. Finally, cells were fixed with FoxP3/Transcription Factor Fixation/Permeabilization kit (eBioscience, San Diego, CA, USA), stained with antibodies against CD4, CD25, and IFN- and analyzed on LSR II flow cytometer. RNA isolation and real-time PCR Total RNA was isolated from cells using RNeasy Plus Micro kit (Qiagen). RNA was quantified, and reverse-transcribed using Superscript III Reverse transcriptase kit (Invitrogen). Real-time PCR was performed using mouse IDO1 primers (forward: 5-TGG CAA ACT GGA AGA AAA AG-3; reverse: 5-AAT GCT TTC AGG TCT TGA CG-3) and mouse Aldh1A2 primers (forward: 5-TGG GTG AGT TTG GCT TAC GG-3; reverse: 5-AGA AAC GTG GCA GTC TTG GC-3). All PCR data were normalized to the data for mouse HPRT1 (primers: 5-GCG TCG TGA.In addition to these extracellular actions of SCFAs, intracellular actions also play an essential role in the biological effects of these bacterial metabolites. 12 h day-night cycles, with water and food provided ad libitum, and used between 8 C 12 weeks of age. Germ-free and age-matched conventional mice were obtained from Taconic Biosciences, Inc. (Hudson, NY, USA) and used as described previously [23]. Institutional Animal Care and CP-547632 Use Committee (IACUC) of the Georgia Regents University approved all animal procedures reported in this study. Isolation of DCs and their culture Mature DCs were isolated from mouse spleen using CD11c microbeads (Miltenyi Biotech, Auburn, CA, USA) followed by magnetic separation. Purity of isolated DCs was determined by flow cytometric analysis using CD11c antibody. DC purity for typical DC isolation was ~90%. DCs were then cultured in complete culture medium (RPMI 1640 medium, Rabbit polyclonal to PDGF C containing 10% fetal calf serum, 10 mM HEPES pH 7.4, 2 mM glutamine, 100 IU/ml penicillin, 100 g/ml streptomycin, and 50 M 2-mercaptoethanol) for 48 h. Isolation and culture of CD4+ T cells CD4+ CD25? CD44+ CD62LHi T cells were isolated from spleen and mesenteric lymph nodes of OT-II transgenic mice by FACS using fluorochrome-conjugated antibodies (both from eBioscience, San Diego, CA, USA). For in vitro FoxP3+ CD4+ Treg conversion assay, DCs pretreated with or without butyrate were recovered from culture at the end of 48 h and co-cultured for 4 days with these OT-II T cells at a ratio 1:2 in complete medium. The culture medium was supplemented with 0.5 g/ml Ovalbumin peptide (ISQVHAAHAEINEA), 0.4 ng/ml TGF and 5 ng/ml IL-2. The cells were then fixed with FoxP3/Transcription Factor Fixation/Permeabilization kit (eBioscience, San Diego, CA, USA) and stained with antibodies against CD4 and FoxP3 for analysis CP-547632 on LSR II flow cytometer. For in vitro IFN-+ CD4+ T cell suppression assay, DCs pretreated with or without butyrate were recovered from culture at the end of 48 h and co-cultured for 4 days with the CD4+ CD25?CD44? CD62LHi there T cells from OT-II transgenic mice at DC:T cell percentage 1:2 in the complete medium. The tradition medium was supplemented with 0.5 g/ml Ovalbumin peptide (ISQVHAAHAEINEA), 10 ng/ml IL-12, 10 g/ml anti-IL4, and 5 ng/ml IL-2. On day time 4 of co-culture, cells were recovered and cultured further in presence of 5 ng/ml IL-2 for 48 h, after which the cells were stimulated with phorbol myristate acetate plus ionomycin in the presence of GolgiStop and Golgiplug for 5 h. Finally, cells were fixed with FoxP3/Transcription Element Fixation/Permeabilization kit (eBioscience, San Diego, CA, USA), stained with antibodies against CD4, CD25, and IFN- and analyzed on LSR II circulation cytometer. In vivo IFN-+ CD4+ T cell suppression assay OVA-specific CD4+ CD25?CD44? CD62LHiCD90.1+ (Thy1.1) T cells from OT-II donor mice were injected (i.v.) into recipient WT or (Thy1.2) CP-547632 mice 1 day before immunization. Mice were then immunized with a mixture of ovalbumin dissolved in PBS and total Freuds adjuvant at 1:1 percentage (s.c.). Two weeks later, animals were sacrificed to obtain cells from spleen. These cells were then stimulated with phorbol myristate acetate plus ionomycin in the presence of GolgiStop and Golgiplug for 5 h. Finally, cells were fixed with FoxP3/Transcription Element Fixation/Permeabilization kit (eBioscience, San Diego, CA, USA), stained with antibodies against CD4, CD25, and IFN- and analyzed on LSR II circulation cytometer. RNA isolation and real-time PCR Total RNA was isolated from cells using RNeasy Plus Micro kit (Qiagen). RNA was quantified, and reverse-transcribed using Superscript III Reverse transcriptase kit (Invitrogen). Real-time PCR was performed using mouse IDO1 primers (ahead: 5-TGG CAA Take action GGA AGA AAA AG-3; opposite: 5-AAT GCT TTC AGG TCT TGA CG-3) and mouse Aldh1A2 primers (ahead: 5-TGG GTG AGT TTG GCT TAC GG-3; opposite: 5-AGA AAC GTG GCA GTC TTG GC-3). All PCR data were normalized to the data for mouse HPRT1 (primers: 5-GCG TCG TGA TTA GCG ATG ATG AAC-3 and 5-CCT CCC ATC TCC.