Journal: Blood

Article Title: Quantitative analysis of murine terminal erythroid differentiation in vivo: novel method to study normal and disordered erythropoiesis

doi: 10.1182/blood-2012-09-456079

Figure Lengend Snippet: Flow cytometric analysis and isolation of erythroblasts of wild-type mice bone marrow cells. (A) Plot of FSC versus SSC of all the cells. (B) Plot of 7AAD versus SSC of all the cells. (C) Plot of CD45CD11bGR1 versus SSC of all the cells. (D) Histogram of TER119 of CD45−CD11b−GR1− cells. (E) Plot of CD44 versus TER119 of the TER119 positive cells without gating. (F) Plot of CD44 versus TER119 of the TER119 positive cells with the gating on CD44hiTERlow population I. (G) Plot of CD44 versus FSC of the TER119 positive cells, showing naturally occurring clusters. (H) Plot of CD44 versus FSC of the TER119 positive cells with gating the population II, III, IV, V and VI. (I) Representative cytospin images of the sorted populations. (J) Histogram of FSC of distinct stage of erythroblasts. (K) Proportion of distinct stage of erythroblasts in bone marrow.

Article Snippet: 17 Antibodies CD16/CD32 (Fcγ III/II Receptor), FITC-conjugated anti-Ter119, APC-conjugated anti-CD44, APC-Cy 7–conjugated anti-CD45, APC-Cy 7–conjugated anti-CD11b, APC-Cy 7–conjugated anti-GR1, and 7AAD were purchased from BD Bioscience.

Techniques: Isolation

Journal: Nature Communications

Article Title: Organoids transplantation attenuates intestinal ischemia/reperfusion injury in mice through L-Malic acid-mediated M2 macrophage polarization

doi: 10.1038/s41467-023-42502-0

Figure Lengend Snippet: A Flow cytometry analysis of neutrophils (CD45 + CD11b + Ly6G + ), monocytes (CD45 + CD11b + Ly6C + ), macrophages (CD45 + CD11b + F4/80 + ), and T cells (CD45 + CD11b - CD3 + ) in the lamina propria (LP) of the small intestine (n = 5 mice for sham group with organoids transplanted, control group at 6 h, transplanted group at 6 h, control group at 12 h, transplanted group at 48 h, n = 6 mice for transplanted group at 12 h and control group at 48 h, n = 4 mice for the rest groups). Neutrophils: represent significant p value using two-tailed student’s from left to right: 0.0270, 0.0293, 0.0406; monocytes: represent significant * p value = 0.0191 using two-tailed student’s t test at 6 h, represent significant * p value = 0.0173 using two-tailed Mann–Whitney test at 12 h, represent significant * p value = 0.0345 using two-tailed student’s t test at 36 h; macrophages: represent significant * p value = 0.0297 using two-tailed student’s t test. B Expression of Ly6C and MHCII by live CD45 + CD11b + small intestinal LP cells in organoid-transplanted and control mice 36 h after intestinal I/R and transplantation. C Expression of CX3CR1 and CD206 in the indicated cell subsets from organoid-transplanted mice. D Frequency of Ly6C + MHCII - , Ly6C + MHCII + , and Ly6C - MHCII + subsets among CD45 + CD11b + cells in the small intestine of different groups (n = 6 mice/group). Ly6C + MHCII - cells: represent significant ** p value = 0.0022 using two-tailed Mann–Whitney test; Ly6C + MHCII + cells: represent significant *** p value = 0.0008 using two-tailed student’s t test; Ly6C - MHCII + cells: represent significant ** p value = 0.0084 using two-tailed student’s t test. E Representative flow cytometric analysis of CD206 + cells in the small intestine LP 36 h after intestinal I/R injury. F Quantification of CD206 + F4/80 + CD45 + CD11b + macrophages (n = 6 mice/group). Represent significant **** p value <0.0001 using two-tailed student’s t test. G Representative histograms of CD206 expression. H Quantification of CD206 mean fluorescence intensity (MFI) in the LP of the small intestine 36 h after intestinal I/R injury (n = 6 mice/group). Represent significant *** p value = 0.0001 using two-tailed student’s t test. I qRT-PCR analysis of CD206 mRNA in F4/80 + macrophages (n = 6 mice/group). Represent significant *** p value = 0.0008 using two-tailed student’s t test. J Quantification of IL-10 + F4/80 + CD45 + CD11b + macrophages (n = 6 mice/group). Represent significant ** p value = 0.0022 using two-tailed Mann–Whitney test. K Representative histograms of IL-10 expression. L Quantification of IL-10 MFI in the small intestine LP 36 h after intestinal I/R injury (n = 6 mice/group). Represent significant ** p value = 0.0022 using two-tailed Mann–Whitney test. M qRT-PCR analysis of IL-10 mRNA expression in F4/80 + macrophages (n = 6 mice/group). Represent significant ** p value = 0.0022 using two-tailed Mann–Whitney test. The statistical tests employed included: two-tailed student’s t test and Mann–Whitney tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Each dot represents data from a single mouse ([ A ], [ D ], [ F ], [ H – J ] and [ L , M ]). Bar graphs represent mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Small intestinal macrophages were cultured in 10% fetal bovine serum (FBS) and stained with APC-Cy7–conjugated anti-CD45 (I3/2.3, A15395, Thermo Fisher Scientific), BV510-conjugated anti-mouse CD11b antibody (clone M1/70, 101263, Biolegend), FITC-conjugated mouse F4/80 (clone BM8, 123108, Biolegend), and sorted.

Techniques: Flow Cytometry, Two Tailed Test, MANN-WHITNEY, Expressing, Transplantation Assay, Fluorescence, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Organoids transplantation attenuates intestinal ischemia/reperfusion injury in mice through L-Malic acid-mediated M2 macrophage polarization

doi: 10.1038/s41467-023-42502-0

Figure Lengend Snippet: A LP macrophages were analyzed using flow cytometry for F4/80 + CD45 + CD11b + cells in mice treated with clodronate liposomes or PBS liposomes 36 h after intestinal I/R injury and transplantation. Representative images and quantification of the macrophage population (n = 5 mice/group). Represent significant p value using two-tailed Mann–Whitney test. 0.0079 (PBS + Lipo, Ctrl vs Org trans), 0.0079 (Org trans, PBS + Lipo vs Clodronate + Lipo). B Hematoxylin and eosin (H & E) staining of small intestinal tissue from mice 36 h after induction of intestinal I/R and quantification of small intestinal pathology score (n = 5 mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0326 (PBS + Lipo, Ctrl vs Org trans), 0.0036 (Org trans, PBS + Lipo vs Clodronate + Lipo). C ELISA detection of IL-6 and IL-1β, and IL-10 production in mice 36 h after intestinal I/R and transplantation (n = 5 mice/group). Represent significant p value using two-tailed Mann–Whitney test for IL-6. 0.0079 (PBS + Lipo, Ctrl vs Org trans), 0.0079 (Org trans, PBS + Lipo vs Clodronate + Lipo). Represent significant p value using two-way ANOVA followed by the Tukey test for IL-1β. 0.0334 (PBS + Lipo, Ctrl vs Org trans), 0.0021 (Org trans, PBS + Lipo vs Clodronate + Lipo). Represent significant p value using two-tailed Mann–Whitney test for IL-10. 0.0079 (PBS + Lipo, Ctrl vs Org trans), 0.0079 (Org trans, PBS + Lipo vs Clodronate + Lipo). D qRT-PCR analysis of Occludin and ZO-1 mRNA in the small intestinal tissue of mice that underwent intestinal I/R and transplantation for 36 h (n = 5 mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test for Occludin and ZO-1. For Occludin, 0.0011 (PBS + Lipo, Ctrl vs Org trans), 0.0004 (Org trans, PBS + Lipo vs Clodronate + Lipo). For ZO-1, <0.0001 (PBS + Lipo, Ctrl vs Org trans), < 0.0001 (Org trans, PBS + Lipo vs Clodronate + Lipo). E Representative immunohistochemistry images of Occludin staining and quantification of the Occludin staining area (n = 5 mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0255 (PBS + Lipo, Ctrl vs Org trans), 0.0042 (Org trans, PBS + Lipo vs Clodronate + Lipo). F Representative immunohistochemistry images of ZO-1 and quantification of the ZO-1 staining area (n = 5 mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0040 (PBS + Lipo, Ctrl vs Org trans), 0.0022 (Org trans, PBS + Lipo vs Clodronate + Lipo). G Expression of Ly6C and MHCII by live CD45 + CD11b + small intestinal LP cells in mice 36 h after intestinal I/R and transplantation. H Frequency of Ly6C + MHCII - , Ly6C + MHCII + , and Ly6C - MHCII + subsets among CD45 + CD11b + cells in the small intestine of different groups (n = 5 mice/group). Represent significant p value using two-tailed Mann–Whitney test for Ly6C + MHCII - , Ly6C + MHCII + , and Ly6C - MHCII + cells. For Ly6C + MHCII - cells, 0.0079 (PBS + Lipo, Ctrl vs Org trans), 0.0079 (Org trans, PBS + Lipo vs Clodronate + Lipo). For Ly6C + MHCII + cells, 0.0079 (Org trans, PBS + Lipo vs Clodronate + Lipo). For Ly6C - MHCII + cells, 0.0159 (PBS + Lipo, Ctrl vs Org trans), 0.0079 (Org trans, PBS + Lipo vs Clodronate + Lipo). I Quantification of CD206 + F4/80 + CD45 + CD11b + macrophages ( n = 5 mice/group). Represent significant p value using two-tailed Mann–Whitney test. 0.0159 (PBS + Lipo, Ctrl vs Org trans), 0.0079 (Org trans, PBS + Lipo vs Clodronate + Lipo). J Representative histograms of CD206 expression. K Quantification of CD206 MFI in the LP of the small intestine 36 h after intestinal I/R injury (n = 5 mice/group). Represent significant p value using two-tailed Mann–Whitney test. 0.0079 (PBS + Lipo, Ctrl vs Org trans), 0.0079 (Org trans, PBS + Lipo vs Clodronate + Lipo). Scale bar, 100 μm. The statistical tests employed included: two-way ANOVA followed by the Tukey test for multiple comparisons and Mann–Whitney tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Each dot represents data from a single mouse ([ A – F ], [ H , I ], and [ K ]). Bar graphs represent mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Small intestinal macrophages were cultured in 10% fetal bovine serum (FBS) and stained with APC-Cy7–conjugated anti-CD45 (I3/2.3, A15395, Thermo Fisher Scientific), BV510-conjugated anti-mouse CD11b antibody (clone M1/70, 101263, Biolegend), FITC-conjugated mouse F4/80 (clone BM8, 123108, Biolegend), and sorted.

Techniques: Flow Cytometry, Liposomes, Transplantation Assay, Two Tailed Test, MANN-WHITNEY, Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Immunohistochemistry, Expressing

Journal: Nature Communications

Article Title: Organoids transplantation attenuates intestinal ischemia/reperfusion injury in mice through L-Malic acid-mediated M2 macrophage polarization

doi: 10.1038/s41467-023-42502-0

Figure Lengend Snippet: A H & E staining of small intestinal tissue from mice 36 h after induction of intestinal I/R and quantification of the small intestinal pathology score (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0167 (Vehicle, Ctrl vs Org trans), 0.0172 (Ctrl, Vehicle vs Malic acid). B qRT-PCR analysis of Occludin and ZO-1 mRNA in small intestinal tissues from mice 36 h after intestinal I/R (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-tailed Mann–Whitney test for Occludin. 0.0043 (Vehicle, Ctrl vs Org trans), 0.0043 (Ctrl, Vehicle vs Malic acid). Represent significant p value using two-way ANOVA followed by the Tukey test for ZO-1. 0.0105 (Vehicle, Ctrl vs Org trans), 0.0259 (Ctrl, Vehicle vs Malic acid). C Representative immunohistochemistry images of Occludin expression and quantification of the area of Occludin immunohistochemical staining (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-tailed Mann–Whitney test. 0.0043 (Vehicle, Ctrl vs Org trans), 0.0022 (Ctrl, Vehicle vs Malic acid). D Representative immunohistochemistry images of ZO-1 and quantification of the area of ZO-1 staining (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-tailed Mann–Whitney test. 0.0043 (Vehicle, Ctrl vs Org trans), 0.0022 (Ctrl, Vehicle vs Malic acid). E ELISA detection of IL-6 and IL-1β production in mice 36 h after intestinal I/R (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. For IL-6, 0.0297 (Vehicle, Ctrl vs Org trans), 0.0190 (Ctrl, Vehicle vs Malic acid). For IL-1β, < 0.0001 (Vehicle, Ctrl vs Org trans), 0.0002 (Ctrl, Vehicle vs Malic acid). F Quantification of CD206 + F4/80 + CD45 + CD11b + macrophages (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-tailed Mann–Whitney test. 0.0043 (Vehicle, Ctrl vs Org trans), 0.0022 (Ctrl, Vehicle vs Malic acid). G Representative histograms of CD206 expression. H Quantification of CD206 MFI in the LP of the small intestine 36 h after intestinal I/R injury (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0056 (Vehicle, Ctrl vs Org trans), 0.0012 (Ctrl, Vehicle vs Malic acid). I Representative histograms of IL-10 expression. J Quantification of IL-10 + F4/80 + CD45 + CD11b + macrophages (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0083 (Vehicle, Ctrl vs Org trans), 0.0085 (Ctrl, Vehicle vs Malic acid). K Quantification of IL-10 MFI in the LP of the small intestine 36 h after intestinal I/R (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-tailed Mann–Whitney test. 0.0022 (Ctrl, Vehicle vs Malic acid). L Serum concentration of IL-10 in mice 36 h after intestinal I/R (n = 5 mice for transplanted groups, n = 6 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0247 (Vehicle, Ctrl vs Org trans), 0.0013 (Ctrl, Vehicle vs Malic acid). M Genetic profiling of BMDMs stimulated with or without MA (n = 4 biological replicates for vehicle group, n = 6 biological replicates for MA group). Represent significant p value using two-tailed student’s from left to right: 0.0108, 0.0096, 0.0015. N Representative images of CD206 expression and quantification of CD206 + F4/80 + CD11b + in BMDMs stimulated with or without MA supplementation (n = 3 biological replicates/group). Represent significant * p value = 0.0266 using two-tailed student’s t test. O Representative images of immunostaining of CD206 (green) and DAPI (blue) in BMDMs stimulated with or without MA supplementation. Scale bar, 100 μm. The statistical tests employed included: two-way ANOVA followed by the Tukey test for multiple comparisons, two-tailed student’s t test and Mann–Whitney tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Each dot represents data from a single sample ([ A – F ], [ H ], and [ J – N ]). Bar graphs represent mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Small intestinal macrophages were cultured in 10% fetal bovine serum (FBS) and stained with APC-Cy7–conjugated anti-CD45 (I3/2.3, A15395, Thermo Fisher Scientific), BV510-conjugated anti-mouse CD11b antibody (clone M1/70, 101263, Biolegend), FITC-conjugated mouse F4/80 (clone BM8, 123108, Biolegend), and sorted.

Techniques: Staining, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Immunohistochemistry, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, Immunostaining

Journal: Nature Communications

Article Title: Organoids transplantation attenuates intestinal ischemia/reperfusion injury in mice through L-Malic acid-mediated M2 macrophage polarization

doi: 10.1038/s41467-023-42502-0

Figure Lengend Snippet: A Gene ontology (GO) pathway enrichment analyses of the upregulated DEGs between two groups. The size of the node represents the number of DEGs, and the color of the node represents the corresponding P value. B Scatter plots comparing the DEGs between the MA administrated and vehicle groups. Genes were monitored according to their expression levels (log10 intensity). Red and green dots represented upregulated and downregulated genes, respectively. C Genetic profiling of SOCS family genes in BMDMs administrated with or without MA. D Representative histograms and quantification of CD206 expression in BMDMs depleted of SOCS2 using siRNA and co-cultured with or without MA (n = 3 biological replicates for siNC administrated vehicle group, n = 4 biological replicates for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0034 (siNC, Vehicle vs MA), 0.0007 (MA, siNC vs siSOCS2). E Representative images of immunostaining of CD206 (green) and DAPI (blue) in BMDMs depleted of SOCS2 using siRNA and co-cultured with or without MA. F Representative histograms of IL-10 expression and quantification of IL-10 + MFI (n = 3 biological replicates/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0335 (siNC, Vehicle vs MA), 0.0072 (MA, siNC vs siSOCS2). G Quantification of IL-10 + F4/80 + CD45 + CD11b + macrophages in BMDMs depleted of SOCS2 using siRNA and co-cultured with or without MA (n = 3 biological replicates/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0353 (siNC, Vehicle vs MA), 0.0066 (MA, siNC vs siSOCS2). H ELISA detection of IL-10 concentration released by BMDMs depleted of SOCS2 using siRNA and co-cultured with or without MA (n = 3 biological replicates/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0020 (siNC, Vehicle vs MA), 0.0001 (MA, siNC vs siSOCS2). Scale bar, 100 μm. The statistical tests employed included: two-way ANOVA followed by the Tukey test for multiple comparisons and two-tailed student’s t test. * P < 0.05, ** P < 0.01, *** P < 0.001. Each dot represents data from a single sample ([ D ], [ F – H ]). Bar graphs represent mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Small intestinal macrophages were cultured in 10% fetal bovine serum (FBS) and stained with APC-Cy7–conjugated anti-CD45 (I3/2.3, A15395, Thermo Fisher Scientific), BV510-conjugated anti-mouse CD11b antibody (clone M1/70, 101263, Biolegend), FITC-conjugated mouse F4/80 (clone BM8, 123108, Biolegend), and sorted.

Techniques: Expressing, Cell Culture, Immunostaining, Enzyme-linked Immunosorbent Assay, Concentration Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Organoids transplantation attenuates intestinal ischemia/reperfusion injury in mice through L-Malic acid-mediated M2 macrophage polarization

doi: 10.1038/s41467-023-42502-0

Figure Lengend Snippet: A H & E staining of small intestinal tissue from mice 36 h after induction of intestinal I/R and quantification of the small intestinal pathology score (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0241 (WT, I/R + Vehicle vs I/R + MA), 0.0020 (I/R + MA, WT vs SOCS2 –/– ). B Quantification of the area of Occludin, ZO-1 immunohistochemical staining (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. For Occludin, 0.0231 (WT, I/R + Vehicle vs I/R + MA), 0.0019 (I/R + MA, WT vs SOCS2 –/– ); for ZO-1, < 0.0001 (WT, I/R + Vehicle vs I/R + MA), < 0.0001 (I/R + MA, WT vs SOCS2 –/– ). C qRT-PCR analysis of Occludin and ZO-1 mRNA in small intestinal tissues from mice 36 h after intestinal I/R (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. For Occludin, 0.0056 (WT, I/R + Vehicle vs I/R + MA), 0.0005 (I/R + MA, WT vs SOCS2 –/– ); for ZO-1, 0.0014 (WT, I/R + Vehicle vs I/R + MA), 0.0007 (I/R + MA, WT vs SOCS2 –/– ). D ELISA detection of IL-6, IL-1β and IL-10 production in mice 36 h after intestinal I/R (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. For IL-6, 0.046 (WT, I/R + Vehicle vs I/R + MA), 0.0046 (I/R + MA, WT vs SOCS2 –/– ); for IL-1β, 0.0159 (WT, I/R + Vehicle vs I/R + MA), 0.0052 (I/R + MA, WT vs SOCS2 –/– ); for IL-10, 0.0005 (WT, I/R + Vehicle vs I/R + MA), 0.0003 (I/R + MA, WT vs SOCS2 –/– ). E Frequency of Ly6C + MHCII - , Ly6C + MHCII + , and Ly6C - MHCII + subsets among CD45 + CD11b + cells in the small intestine of different groups (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test for Ly6C + MHCII - and Ly6C + MHCII + cells. For Ly6C + MHCII - cells, 0.0169 (WT, I/R + Vehicle vs I/R + MA), 0.0221 (I/R + MA, WT vs SOCS2 –/– ); for Ly6C + MHCII + cells, 0.0004 (WT, I/R + Vehicle vs I/R + MA), 0.0011 (I/R + MA, WT vs SOCS2 –/– ). F Quantification of CD206 + F4/80 + CD45 + CD11b + macrophages (n = 3 in SOCS2 –/– mice/group, n = 4 in WT mice/group). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0004 (WT, I/R + Vehicle vs I/R + MA), 0.0012 (I/R + MA, WT vs SOCS2 –/– ). G Representative histograms of CD206 expression in the LP of the small intestine 36 h after intestinal I/R injury. H Schematic illustration of the induction protocols for macrophage adoptive transfer. I H & E staining of small intestinal tissue from mice 36 h after induction of macrophages adoptive transfer and quantification of the small intestinal pathology score (n = 3 for recipient SOCS2 -/- mice adoptive transfer of the macrophages from MA-administrated WT mice group, n = 4 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. 0.0163 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0258 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages). J Quantification of the area of Occludin, ZO-1 immunohistochemical staining (n = 3 for recipient SOCS2 -/- mice adoptive transfer of the macrophages from MA-administrated WT mice group, n = 4 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. For Occludin, 0.0003 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0023 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages); for ZO-1, 0.0018 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0033 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages). K ELISA detection of IL-6, IL-1β and IL-10 production in mice 36 h after macrophages adoptive transfer (n = 3 for recipient SOCS2 -/- mice adoptive transfer of the macrophages from MA-administrated WT mice group, n = 4 mice for the rest groups). Represent significant p value using two-way ANOVA followed by the Tukey test. For IL-6, 0.0288 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0226 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages); for IL-1β, 0.0012 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0004 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages); for IL-10, 0.0022 (WT, Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages), 0.0154 (SOCS2 –/– , Adoptive transfer of WT macrophages vs Adoptive transfer of SOCS2 –/– macrophages). Scale bar, 200 μm. The statistical tests employed included: two-way ANOVA followed by the Tukey test for multiple comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Each dot represents data from a single sample ([ A – F ], [ I – K ]). Bar graphs represent mean ± SD. Source data are provided as a Source Data file.

Article Snippet: Small intestinal macrophages were cultured in 10% fetal bovine serum (FBS) and stained with APC-Cy7–conjugated anti-CD45 (I3/2.3, A15395, Thermo Fisher Scientific), BV510-conjugated anti-mouse CD11b antibody (clone M1/70, 101263, Biolegend), FITC-conjugated mouse F4/80 (clone BM8, 123108, Biolegend), and sorted.

Techniques: Staining, Immunohistochemistry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Adoptive Transfer Assay

Journal: PLoS ONE

Article Title: Xenogeneic Graft-versus-Host-Disease in NOD- scid IL-2Rγ null Mice Display a T-Effector Memory Phenotype

doi: 10.1371/journal.pone.0044219

Figure Lengend Snippet: Peripheral blood was collected from Hu-PBMC mice bi-weekly and stained with anti-human specific antibodies for human (A) CD45, (B) CD3, (C) CD20, (D) CD25, and (E) CD45RO and quantified by flow cytometry. 2-way ANOVA test was performed. Each data point represents the average ( n = 4 NSG and BRG mice respectively, 2 PBMC donors) ±SEM values.

Article Snippet: The following antibodies were used in dilutions according to manufacturers instructions: FITC conjugated anti-human CD62L (Invitrogen), PE conjugated anti-human CD27 (eBioscience), PE conjugated anti-human CD25 (BD), PE conjugated anti-human CLA (Miltenyi), PE-Cy5.5 conjugated anti-human CD3 (Invitrogen), PE-Cy5.5 conjugated anti-human CD20 (Invitrogen), PE-Cy7 conjugated anti-mouse CD45 (eBioscience), PE-TR conjugated anti-human CD4 (Invitrogen), APC conjugated anti-human CD8 (Invitrogen), APC-Cy7 conjugated anti-human CD45 (eBioscience), and Pacific Blue conjugated anti-human CD45RO (BD).

Techniques: Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Xenogeneic Graft-versus-Host-Disease in NOD- scid IL-2Rγ null Mice Display a T-Effector Memory Phenotype

doi: 10.1371/journal.pone.0044219

Figure Lengend Snippet: Cells were harvested from the spleens of Hu-PBMC NSG and BRG mice at weekly intervals following transfer and stained with anti-human specific antibodies for mouse CD45 and for human CD45, CD3, CD4, and CD20. Samples were analysed by flow cytometry to calculate the % of human (A) CD45 + , (C) CD3 + and (G) CD20 + , the absolute accumulation of human (B) CD45 + , (D) CD3 + , (E) CD8 + , (F) CD4 + and (H) CD20 + cells. 2-way ANOVA test was performed. Data are compiled from 2 independent experiments (NSG n = 4–5 per time point and BRG n = 4 day 7–21 or n = 2 day 28, 2 PBMC donors).

Article Snippet: The following antibodies were used in dilutions according to manufacturers instructions: FITC conjugated anti-human CD62L (Invitrogen), PE conjugated anti-human CD27 (eBioscience), PE conjugated anti-human CD25 (BD), PE conjugated anti-human CLA (Miltenyi), PE-Cy5.5 conjugated anti-human CD3 (Invitrogen), PE-Cy5.5 conjugated anti-human CD20 (Invitrogen), PE-Cy7 conjugated anti-mouse CD45 (eBioscience), PE-TR conjugated anti-human CD4 (Invitrogen), APC conjugated anti-human CD8 (Invitrogen), APC-Cy7 conjugated anti-human CD45 (eBioscience), and Pacific Blue conjugated anti-human CD45RO (BD).

Techniques: Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Xenogeneic Graft-versus-Host-Disease in NOD- scid IL-2Rγ null Mice Display a T-Effector Memory Phenotype

doi: 10.1371/journal.pone.0044219

Figure Lengend Snippet: Peripheral blood, spleen, lymph nodes, and bone marrow were harvested at GvHD development in Hu-PBMC NSG and BRG mice and stained with anti-human specific antibodies for human (A) CD45, (B) CD3, (C) CD4, (D) CD25, (E) CD45RO, and (F) CD20 and quantified by flow cytometry. Tukey box-and-whisker graphs are shown. Unpaired t test was performed. Data are compiled from 3 independent experiments ( n = 4 NSG and BRG mice respectively, 2 PBMC donors).

Article Snippet: The following antibodies were used in dilutions according to manufacturers instructions: FITC conjugated anti-human CD62L (Invitrogen), PE conjugated anti-human CD27 (eBioscience), PE conjugated anti-human CD25 (BD), PE conjugated anti-human CLA (Miltenyi), PE-Cy5.5 conjugated anti-human CD3 (Invitrogen), PE-Cy5.5 conjugated anti-human CD20 (Invitrogen), PE-Cy7 conjugated anti-mouse CD45 (eBioscience), PE-TR conjugated anti-human CD4 (Invitrogen), APC conjugated anti-human CD8 (Invitrogen), APC-Cy7 conjugated anti-human CD45 (eBioscience), and Pacific Blue conjugated anti-human CD45RO (BD).

Techniques: Staining, Flow Cytometry, Whisker Assay

Journal: PLoS ONE

Article Title: Xenogeneic Graft-versus-Host-Disease in NOD- scid IL-2Rγ null Mice Display a T-Effector Memory Phenotype

doi: 10.1371/journal.pone.0044219

Figure Lengend Snippet: 10 7 freshly isolated PBMCs were injected intravenously via the tail vein into adult 6–12 week old NSG immunodeficient mice 24 hours post sub lethal irradiation (2.4 Gys). (A) Survival of irradiated Hu-PBMC NSG mice (MST = 14 days, n = 7, 2 PBMC donors). (B) The average weight is shown as a percentage of starting weight. Peripheral blood, spleen and bone marrow were harvested from irradiated Hu-PBMC NSG mice at the indicated time points and analysed for (C) human CD45 engraftment and (D) CD4:CD8 ratio. Black bar represents human PBMC phenotype pre-injection. Data represent the mean ±SEM and are compiled from 2 independent experiments (n = 7). (A) Log-rank Mantel-Cox Test, (B, D) unpaired t test, and (C) 2-way ANOVA, spleen vs blood (*,**) and spleen vs bone marrow (##).

Article Snippet: The following antibodies were used in dilutions according to manufacturers instructions: FITC conjugated anti-human CD62L (Invitrogen), PE conjugated anti-human CD27 (eBioscience), PE conjugated anti-human CD25 (BD), PE conjugated anti-human CLA (Miltenyi), PE-Cy5.5 conjugated anti-human CD3 (Invitrogen), PE-Cy5.5 conjugated anti-human CD20 (Invitrogen), PE-Cy7 conjugated anti-mouse CD45 (eBioscience), PE-TR conjugated anti-human CD4 (Invitrogen), APC conjugated anti-human CD8 (Invitrogen), APC-Cy7 conjugated anti-human CD45 (eBioscience), and Pacific Blue conjugated anti-human CD45RO (BD).

Techniques: Isolation, Injection, Irradiation

Journal: Mucosal immunology

Article Title: Inhibition of CD23-mediated IgE transcytosis suppresses the initiation and development of allergic airway inflammation

doi: 10.1038/mi.2015.16

Figure Lengend Snippet: A. Mice were sensitized (i.p.) with OVA or left untreated. Lung and trachea epithelial cells were isolated and cell lysates (50 µg) were subjected to 12 % SDS-PAGE gel electrophoresis under reducing conditions. Proteins were transferred onto nitrocellulose membranes and blotted with rat-anti mouse CD23 mAb (B3B4) followed by HRP-conjugated rabbit anti-rat IgG Ab. Proteins were visualized using ECL. Arrows indicate mouse CD23 and β-tubulin. B–D. WT/WT, CD23KO/WT, WT/CD23KO chimeric mice were i.p. sensitized with 100 µg OVA plus 4 mg alum at day 0 and subsequently injected (i.p.) with 100 µg OVA on day 7 and 14. Mice were challenged on day 21 with nebulized 1 % OVA for 30 min. 5 h after OVA challenge, sera were collected and OVA quantitated by ELISA (B). 24 h after OVA challenge, BAL fluid and sera were collected and OVA-specific IgE (C) and IL-4 (D) concentrations assessed by ELISA. *P<0.05. E. Flow cytometric analysis of eosinophils in BAL fluid. Chimeric mice were sensitized with OVA and received a single aerosol OVA challenge. Total leukocytes obtained from BAL fluid were gated on CD45 + CD11b hi/int and mononuclear cells obtained from lung were gated on CD45 + CD11b hi cells and further analyzed for CD11c and Siglec-F expression (E1). The mean percentage of eosinophils in BAL fluid from three different experiments was calculated (E2). *P<0.05. F. Airway responsiveness to methacholine (MCh) in chimeric and control mice . WT/WT, CD23KO/WT, and WT/CD23KO chimeric mice were sensitized (i.p.) with OVA and received a single aerosol challenge with OVA while control mice were sensitized and received a single aerosol challenge with PBS. Airway resistance was measured 24 h after challenge. First, the baseline and resistance against PBS challenge were measured followed by generation of a dose-response curve against an increasing concentration of nebulized MCh (3–100 mg/ml). *P<0.05.

Article Snippet: FITC-conjugated rat anti-mouse B3B4 mAb and rat anti-mouse CD16/CD32 Ab, FITC-conjugated hamster anti-mouse CD11c, FITC-conjugated hamster IgG1, PE-conjugated rat anti-mouse Siglec-F, PE-conjugated rat IgG2a, APC-Cy7-conjugated rat anti-mouse CD45, APC-Cy7-conjugated rat IgG2b were obtained from BD Pharmingen (San Diego, CA).

Techniques: Isolation, SDS Page, Nucleic Acid Electrophoresis, Injection, Enzyme-linked Immunosorbent Assay, Expressing, Concentration Assay

Journal: Mucosal immunology

Article Title: Inhibition of CD23-mediated IgE transcytosis suppresses the initiation and development of allergic airway inflammation

doi: 10.1038/mi.2015.16

Figure Lengend Snippet: A. B3B4 Ab blocked IgE transcytosis in primary mouse TEC. Cells were isolated from WT mice, grown on transwell filters, and allowed to polarized. Purified B3B4 Ab or rat IgG2a (50 µg/ml) was added into the apical chamber for 45 min at 4°C to allow Ab binding to apical CD23. Mouse IgE was then added to apical chambers and incubated at 37°C for 2 h. Medium from the basolateral chamber was collected and IgE content measured by ELISA. *P<0.05. B–F. Sensitization of mice with OVA. Before challenge, OVA-sensitized WT mice received i.n. inoculation with 75 µg B3B4 Ab or rat IgG2a in PBS twice, once at 24 h before, and again 1 h before, challenge. Sera were collected 5 h after the single OVA challenge and OVA in sera was quantified by ELISA (B). BAL fluid or sera were collected 48 h after OVA challenge. Concentrations of OVA-specific IgE (C), IL-13 (D), and IL-5 (E) in either BAL fluid or sera were measured by ELISA. Eosinophils were assessed by flow cytometric analysis of CD45 + CD11b hi/int CD11c − Siglec-F + cells in BAL fluid. The mean percentage of the eosinophils in the BAL fluid from three different experiments was calculated (F2). *P<0.05.

Article Snippet: FITC-conjugated rat anti-mouse B3B4 mAb and rat anti-mouse CD16/CD32 Ab, FITC-conjugated hamster anti-mouse CD11c, FITC-conjugated hamster IgG1, PE-conjugated rat anti-mouse Siglec-F, PE-conjugated rat IgG2a, APC-Cy7-conjugated rat anti-mouse CD45, APC-Cy7-conjugated rat IgG2b were obtained from BD Pharmingen (San Diego, CA).

Techniques: Isolation, Purification, Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay