Journal: Regenerative therapy

Article Title: Xenogeneic-free defined conditions for derivation and expansion of human embryonic stem cells with mesenchymal stem cells.

doi: 10.1016/j.reth.2014.12.004

Figure Lengend Snippet: Fig. 4. Pluripotent marker expression of xenogeneic-free SEES cell lines In the undifferentiated state, xenogeneic-free SEES cell lines expressed markers characteristic of pluripotent hESCs, including OCT4, NANOG, SOX2, SSEA4, and TRA1-60. SEES-4: scale bars are 200 mm; SEES-5: scale bars are 50 mm in OCT3/4 and TRA1-60 and 200 mm in SOX2 and SSEA4; SEES-6: scale bars are 50 mm in OCT3/4 and SOX2 and 200 mm in SSEA4; SEES-7: scale bars are 200 mm.

Article Snippet: Antibodies against human SSEA-1 (R&D Systems; #FAB2155C), SSEA4 (R&D Systems; #FAB1435F), TRA1-60 (Merck Millipore; #MAB4360), and TRA1-81 (Merck Millipore; #MAB4381) were used as primary antibodies in hESCs.

Techniques: Marker, Expressing

Journal: Regenerative therapy

Article Title: Xenogeneic-free defined conditions for derivation and expansion of human embryonic stem cells with mesenchymal stem cells.

doi: 10.1016/j.reth.2014.12.004

Figure Lengend Snippet: Fig. 6. Characterization of the pluripotency of SEES-2 maintained using a modified conventional hESC culture medium SEES-2 cells were stably maintained over 20 passages on the qualified MEF feeder layer in the modified medium, which contained pharmaceutical-grade recombinant human bFGF (trafermin) and high-dose (35-K) gamma-irradiated KO-SR without antibiotics. A) Typical hESC colony morphology was readily visible. ALP activity was detected. B) SEES-2 cells expressed undifferentiated hESC markers, including OCT4, NANOG, SOX2, SSEA4, and TRA1-60. SEES-2 cells could differentiate into three embryonic germ layers in vitro and in vivo. Scale bars are 200 mm. C) SEES cells that had been differentiated in vitro via EBs expressed markers of the primary germ layers, ectoderm (TUJ1), mesoderm (aSMA), and endoderm (AFP). Scale bars are 100 mm. D) Histological analysis of teratomas containing multidifferentiated tissues derived from SEES-2 cells. Pigmented epithelium (ectoderm), cartilage (mesoderm), and gut epithelial tissues (endoderm). E) Chromosomal analysis of SEES-2 cells cultivated through 16 passages using a modified conventional hESC culture medium showed a normal 46,XX karyotype.

Article Snippet: Antibodies against human SSEA-1 (R&D Systems; #FAB2155C), SSEA4 (R&D Systems; #FAB1435F), TRA1-60 (Merck Millipore; #MAB4360), and TRA1-81 (Merck Millipore; #MAB4381) were used as primary antibodies in hESCs.

Techniques: Stable Transfection, Recombinant, Irradiation, Activity Assay, In Vitro, In Vivo, Derivative Assay

Journal: Regenerative therapy

Article Title: Xenogeneic-free defined conditions for derivation and expansion of human embryonic stem cells with mesenchymal stem cells.

doi: 10.1016/j.reth.2014.12.004

Figure Lengend Snippet: Fig. 7. Expression of pluripotency markers in SEES-2 cells was maintained using a modified conventional hESC culture medium Flow cytometric analysis of hESC-specific marker expression in SEES-2 cells. The isotype control is indicated by the blue line, and the unlabeled sample, which was used as a control, is indicated by the red line. Surface staining is shown by the yellow line for SSEA4, SSEA1, TRA-1-60, and TRA-1-81.

Article Snippet: Antibodies against human SSEA-1 (R&D Systems; #FAB2155C), SSEA4 (R&D Systems; #FAB1435F), TRA1-60 (Merck Millipore; #MAB4360), and TRA1-81 (Merck Millipore; #MAB4381) were used as primary antibodies in hESCs.

Techniques: Expressing, Marker, Control, Staining

Journal: Frontiers in Immunology

Article Title: Nematode-Infected Mice Acquire Resistance to Subsequent Infection With Unrelated Nematode by Inducing Highly Responsive Group 2 Innate Lymphoid Cells in the Lung

doi: 10.3389/fimmu.2018.02132

Figure Lengend Snippet: Strongyloides venezuelensis -experienced mice develop an increased pulmonary inflammation and a resistance against Nippostrongylus brasiliensis infection. (A) Experimental workflow for sequential nematode infection. S. venezuelensis (Sv)-infected or uninfected mice were inoculated with 500 N. brasiliensis (Nb) L3 at day 28. B6; C57BL/6, WT; wild type, sac; sacrificed. (B) The number of lung stage N. brasiliensis larva. Two days after N. brasiliensis infection, migrating larva were isolated from the lungs and counted ( n = 11). cont, control (uninfected at day 0). Pooled data from two independent experiments are shown (Mean ± SD). (C) The numbers of worms in the intestine were counted at indicated days ( n = 5). (D) The numbers of eggs per gram feces (EPG) from each group at day 7 post- N. brasiliensis infection ( n = 7). (E–G) The numbers of eosinophils and ILC2s (E) , the amounts of IL-5 and IL-13 in the BALF (F) , and the Il5, Il13 , and Il33 mRNA expression levels in the lungs as analyzed by Q-PCR (G) from day 35 are shown ( n = 4–5). uninf.; uninfected at day 28. Statistical analyses were conducted using two-way ANOVAs with Bonferroni post-hoc tests (B,E,G) , Wilcoxon matched-pairs signed rank tests, and two-tailed (C) or unpaired t-tests (F) . Data are representative of two independent experiments. (H) C57BL/6 WT mice ( n = 5–6) were infected with 5000 L3 S. venezuelensis at day 0. The numbers of ILC2s, eosinophils, CD3 + /B220 + cells, and monocytes among the BALF cells at the indicated days were analyzed by flow cytometry (FACScalibur). Cell populations were defined as follows: ILC2s, FSC lo SSC lo Lin(CD3, CD4, CD8, CD19, NK1.1, IgE, Gr-1, siglecF) − Sca-1 + ST2 + ; Eosinophils, CD45 + CD3 − B220 − CCR3 + , CD3/B220: CD45 + CCR3 − CD3 + /B220 + ; and Monocytes, CD45 + Autofluorescence high . Data were compared with day 0 data by one-way ANOVA. Data are representative of two independent experiments.

Article Snippet: Fluorescent-labeled antibodies for CCR3 (FAB729F, FITC, #83101) was purchased from R&D Systems, Siglec F (552126, PE, E50-2440) was purchased from BD Biosciences, for Gr-1 (108408, PE, RB6-8C5), CD45 (103134, brilliant violet 421, 30F11), Thy1.2 (140306, pacific blue, 53-2.1), Sca-1 (108142, AlexaFluor700 or 108112, APC, D7), CD3 (100308, PE or 100306, FITC, 145-2C11), CD4 (100512, PE or 100540, PerCP-Cy5.5, RM4-5), CD8 (100708, PE, 53-6.7), CD11b (101208, PE or 101206, FITC, M1/70), CD19 (115508, PE, 6D5), NK1.1 (108708, PE or 108706, FITC, PK136), B220 (103206, FITC, RA3-6B2), and IL-5 (504306, APC, TRFK5) were purchased from BioLegend, for IL-13 (12-7133-41, PE, eBio13A), GATA3 (12-9966-42, PE, TWAJ), and IL-25R (12-7361-82, PE, MUNC33) were purchased from eBioscience, for T1/ST2 (101001F, FITC or 101001B, biotin, DJ8) was purchased from MD Biosciences, and for IgE (1130-09L, PE, 23G3) was purchased from Southern Biotechnology Associates Inc. PerCP-Cy5.5-streptavidin was purchased from BioLegend (405214).

Techniques: Infection, Isolation, Expressing, Two Tailed Test, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Nematode-Infected Mice Acquire Resistance to Subsequent Infection With Unrelated Nematode by Inducing Highly Responsive Group 2 Innate Lymphoid Cells in the Lung

doi: 10.3389/fimmu.2018.02132

Figure Lengend Snippet: ILC2s are critical for the enhanced inflammation and the protection against Nippostrongylus brasiliensis infection. (A) Experimental workflow for sequential nematode infection in bone marrow chimera mice. BMT; bone marrow transplantation, sac; sacrificed. S. venezuelensis -infected (Sv) or uninfected (cont) mice ( Rora sg/+ , n = 6–7; Rora sg/sg , n = 7) were infected with N. brasiliensis (Nb). Five days after N. brasiliensis infection, (B) ILC2s and eosinophils among the BALF cells were analyzed by flow cytometry (LSRFortessa). Cell populations were defined as follows: ILC2s, FSC lo SSC lo CD45 + CD4 − Lin(CD3, CD8, CD19, NK1.1, IgE, siglec F, Gr-1) − Sca-1 + ST2 + and Eosinophils, CD45 + CD11c lo CD3 − B220 − CCR3 + . (C) Il33, Il5 , and Il13 mRNA expression levels in the lungs were examined. (D) The numbers of worms in the intestine were counted at day 5 post- N. brasiliensis infection. Data are representative of two independent experiments. (E) S. venezuelensis -infected or uninfected WT mice were infected with N. brasiliensis after anti-CD4 Ab treatment (αCD4) as in Figure . The numbers of worms in the intestine were counted at day 5 after N. brasiliensis infection ( n = 10–11). Pooled data from two independent experiments are shown (Mean ± SD).

Article Snippet: Fluorescent-labeled antibodies for CCR3 (FAB729F, FITC, #83101) was purchased from R&D Systems, Siglec F (552126, PE, E50-2440) was purchased from BD Biosciences, for Gr-1 (108408, PE, RB6-8C5), CD45 (103134, brilliant violet 421, 30F11), Thy1.2 (140306, pacific blue, 53-2.1), Sca-1 (108142, AlexaFluor700 or 108112, APC, D7), CD3 (100308, PE or 100306, FITC, 145-2C11), CD4 (100512, PE or 100540, PerCP-Cy5.5, RM4-5), CD8 (100708, PE, 53-6.7), CD11b (101208, PE or 101206, FITC, M1/70), CD19 (115508, PE, 6D5), NK1.1 (108708, PE or 108706, FITC, PK136), B220 (103206, FITC, RA3-6B2), and IL-5 (504306, APC, TRFK5) were purchased from BioLegend, for IL-13 (12-7133-41, PE, eBio13A), GATA3 (12-9966-42, PE, TWAJ), and IL-25R (12-7361-82, PE, MUNC33) were purchased from eBioscience, for T1/ST2 (101001F, FITC or 101001B, biotin, DJ8) was purchased from MD Biosciences, and for IgE (1130-09L, PE, 23G3) was purchased from Southern Biotechnology Associates Inc. PerCP-Cy5.5-streptavidin was purchased from BioLegend (405214).

Techniques: Infection, Transplantation Assay, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Nematode-Infected Mice Acquire Resistance to Subsequent Infection With Unrelated Nematode by Inducing Highly Responsive Group 2 Innate Lymphoid Cells in the Lung

doi: 10.3389/fimmu.2018.02132

Figure Lengend Snippet: Eosinophils are essential for resistance against Nippostrongylus brasiliensis . (A–C) S. venezuelensis -infected (Sv) or uninfected (cont) mice ( n = 5) were treated with anti-IL-5 Ab (TRFK5, αIL-5) or control Ab (cont IgG1) at day 0 and day 2 post-infection with N. brasiliensis (Nb). (A) The numbers of worms in the intestine were counted at day 5 after N. brasiliensis infection. (B) ILC2s, eosinophils, and CD4 + cells among the BALF cells were analyzed by flow cytometry (SP6800). Cell populations were defined as follows: ILC2s, FSC lo SSC lo CD45 + CD4 − Lin(CD3, CD8, CD19, NK1.1, IgE, siglec F, Gr-1) − Sca-1 + ST2 + ; Eosinophils, CD45 + CD11c lo CD3 − B220 − CCR3 + ; and CD4 + cells, FSC lo SSC lo CD45 + CD4 + . (C) Il33, Il13 , and Epx mRNA expression levels in the lungs were examined. Data are representative of two independent experiments that had similar results. (D–F) S. venezuelensis -infected or uninfected (cont) mice (WT, n = 7; Δ dblGATA, n = 5–6) were infected with N. brasiliensis . (D) The numbers of worms in the intestine were counted at day 5 after N. brasiliensis infection. (E) Eosinophils, ILC2s, and CD4 + cells among the BALF cells were analyzed as in (B) . (F) Il33, Il5 , and Il13 mRNA expression levels in the lungs were examined. Data are representative of two independent experiments that had similar results.

Article Snippet: Fluorescent-labeled antibodies for CCR3 (FAB729F, FITC, #83101) was purchased from R&D Systems, Siglec F (552126, PE, E50-2440) was purchased from BD Biosciences, for Gr-1 (108408, PE, RB6-8C5), CD45 (103134, brilliant violet 421, 30F11), Thy1.2 (140306, pacific blue, 53-2.1), Sca-1 (108142, AlexaFluor700 or 108112, APC, D7), CD3 (100308, PE or 100306, FITC, 145-2C11), CD4 (100512, PE or 100540, PerCP-Cy5.5, RM4-5), CD8 (100708, PE, 53-6.7), CD11b (101208, PE or 101206, FITC, M1/70), CD19 (115508, PE, 6D5), NK1.1 (108708, PE or 108706, FITC, PK136), B220 (103206, FITC, RA3-6B2), and IL-5 (504306, APC, TRFK5) were purchased from BioLegend, for IL-13 (12-7133-41, PE, eBio13A), GATA3 (12-9966-42, PE, TWAJ), and IL-25R (12-7361-82, PE, MUNC33) were purchased from eBioscience, for T1/ST2 (101001F, FITC or 101001B, biotin, DJ8) was purchased from MD Biosciences, and for IgE (1130-09L, PE, 23G3) was purchased from Southern Biotechnology Associates Inc. PerCP-Cy5.5-streptavidin was purchased from BioLegend (405214).

Techniques: Infection, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Nematode-Infected Mice Acquire Resistance to Subsequent Infection With Unrelated Nematode by Inducing Highly Responsive Group 2 Innate Lymphoid Cells in the Lung

doi: 10.3389/fimmu.2018.02132

Figure Lengend Snippet: IL-33 is not sufficient for the induction of trained ILC2s. WT C57BL/6 mice were subcutaneously inoculated with 5000 S. venezuelensis L3 at day 0 (Sv) or were intranasally administrated 100 ng of IL-33 at days 0, 1, 2, and 3 (IL-33). cont, untreated control. (A) Cell populations in the lung cells, BALF cells, and peripheral blood leukocytes (PBLs) at indicated days were analyzed by flow cytometry (SP6800) ( n = 4). Cell populations were defined as follows: Eosinophils, PI − CD45 + CD11c int CD3 − B220 − CCR3 + and ILC2s, PI − FSC lo SSC lo CD45 + Thy1.2 + Lin − Sca-1 + ST2 + . (B) Four weeks after treatment, lung cells were stimulated with PMA and ionomycin for 3 h in the presence of brefeldin A. Intracellular levels of IL-5 and IL-13 in ILC2s were analyzed by flow cytometry (SP6800) as in Figure . (C) The proportions of IL-5 + IL-13 − , IL-5 + IL-13 + , and IL-5 − IL-13 + cells within the population of ILC2s ( n = 4). (D,E) Mice were infected with N. brasiliensis 4 weeks after S. venezuelensis infection or IL-33 treatment. Control mice were treated with PBS (PBS) instead of IL-33. Five days after N. brasiliensis infection, (D) the numbers of worms in the intestine were counted ( n = 7). (E) The numbers of ILC2s, eosinophils, and macrophages ( n = 5–7) among the BALF cells were examined. Statistical analyses were performed using a two-way ANOVA (A,C) or one-way ANOVA (D) with Bonferroni post-hoc tests. Data are representative of three independent experiments (Mean ± SD).

Article Snippet: Fluorescent-labeled antibodies for CCR3 (FAB729F, FITC, #83101) was purchased from R&D Systems, Siglec F (552126, PE, E50-2440) was purchased from BD Biosciences, for Gr-1 (108408, PE, RB6-8C5), CD45 (103134, brilliant violet 421, 30F11), Thy1.2 (140306, pacific blue, 53-2.1), Sca-1 (108142, AlexaFluor700 or 108112, APC, D7), CD3 (100308, PE or 100306, FITC, 145-2C11), CD4 (100512, PE or 100540, PerCP-Cy5.5, RM4-5), CD8 (100708, PE, 53-6.7), CD11b (101208, PE or 101206, FITC, M1/70), CD19 (115508, PE, 6D5), NK1.1 (108708, PE or 108706, FITC, PK136), B220 (103206, FITC, RA3-6B2), and IL-5 (504306, APC, TRFK5) were purchased from BioLegend, for IL-13 (12-7133-41, PE, eBio13A), GATA3 (12-9966-42, PE, TWAJ), and IL-25R (12-7361-82, PE, MUNC33) were purchased from eBioscience, for T1/ST2 (101001F, FITC or 101001B, biotin, DJ8) was purchased from MD Biosciences, and for IgE (1130-09L, PE, 23G3) was purchased from Southern Biotechnology Associates Inc. PerCP-Cy5.5-streptavidin was purchased from BioLegend (405214).

Techniques: Flow Cytometry, Infection