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recombinant rat il4 (Beyotime)

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Beyotime recombinant rat il4
Recombinant Rat Il4, supplied by Beyotime, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/recombinant+rat+il4/recombinant+rat+il4/pmc10328259__NRR___18___2208_Suppl1-10-0-5
Average 90 stars, based on 1 article reviews

recombinant rat il4 - by Bioz Stars, 2026-10

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Recombinant:

Article Title:
Article Snippet: .. Recombinant rat IL4 (Cat# P6267, Beyotime Institute of Biotechnology, Nantong, Jiangsu, China) (800 ng/d in 10 μL PBS) was designed to achieve a constant infusion rate of 0.5 μL/h for 7 or 14 days by a micro-infusion pump, starting from 2 hours after MCAO/R. ..

Article Title: M2 macrophages mediate fibrotic scar formation in the early stages after cerebral ischemia in rats.
Article Snippet: .. To do this, BMDMs and RAW264.7 cells were starved for 24 hours by incubation with DMEM containing 2% FBS and subsequently stimulated with or without recombinant rat IL4 (20 ng/mL, Beyotime Institute of Biotechnology, Nantong, Jiangsu, China, Cat# P6267) for 2 days. ..

Incubation:

Image Search Results

HCN channel and KCNQ/Kv7 channel expression on primary microglia. #p < 0.05, ##p < 0.01, ###p < 0.001 compared different experimental groups as marked by horizontal bar. a A representative image of primary microglia culture shows purity by staining of Iba1 (red). Hoechst stains all cell nuclei blue. Images were obtained with a fluorescence microscope, scale bars = 100 μm. b Expression of microglia marker Iba1 and P2RY12. Expression of microglial ion channels KCNK13, the HCN-subunits HCN1, HCN2, HCN3, and HCN4, as well as the KCNQ/Kv7 channel subunits KCNQ2, 3, and 5, on primary rat microglia in vitro measured by RT-qPCR. Microglia were untreated, activated with LPS (10 ng/ml) or IL4 (50 ng/ml; for Iba1: F(2, 6) = 23.571, p = 0.001, ω = 0.913; for P2YR12. F(2, 6) = 31.897, p = 0.001, ω = 0.934; for HCN2: F(2, 18) = 113.981, p < 0.0001, ω = 0.956; for HCN3: F(2, 8) = 22.573, p = 0.001, ω = 0.892; for KCNQ3: F(2, 11) = 11.674, p = 0.002, ω = 0.777; for KCNQ5: H (2) = 9.168, p = 0.01)

Journal: Journal of Neuroinflammation

Article Title: The impact of hyperpolarization-activated cyclic nucleotide-gated (HCN) and voltage-gated potassium KCNQ/Kv7 channels on primary microglia function

doi: 10.1186/s12974-020-01779-4

Figure Lengend Snippet: HCN channel and KCNQ/Kv7 channel expression on primary microglia. #p < 0.05, ##p < 0.01, ###p < 0.001 compared different experimental groups as marked by horizontal bar. a A representative image of primary microglia culture shows purity by staining of Iba1 (red). Hoechst stains all cell nuclei blue. Images were obtained with a fluorescence microscope, scale bars = 100 μm. b Expression of microglia marker Iba1 and P2RY12. Expression of microglial ion channels KCNK13, the HCN-subunits HCN1, HCN2, HCN3, and HCN4, as well as the KCNQ/Kv7 channel subunits KCNQ2, 3, and 5, on primary rat microglia in vitro measured by RT-qPCR. Microglia were untreated, activated with LPS (10 ng/ml) or IL4 (50 ng/ml; for Iba1: F(2, 6) = 23.571, p = 0.001, ω = 0.913; for P2YR12. F(2, 6) = 31.897, p = 0.001, ω = 0.934; for HCN2: F(2, 18) = 113.981, p < 0.0001, ω = 0.956; for HCN3: F(2, 8) = 22.573, p = 0.001, ω = 0.892; for KCNQ3: F(2, 11) = 11.674, p = 0.002, ω = 0.777; for KCNQ5: H (2) = 9.168, p = 0.01)

Article Snippet: Microglia were left unstimulated (control) or were stimulated with 10 ng/ml lipopolysaccharide (LPS derived from E. coli 0111: B4, Sigma Aldrich, St. Louis, USA) to activate microglia towards a pro-inflammatory phenotype, or alternatively stimulated with 50 ng/ml recombinant rat interleukin-4 (IL4; Peprotech, Hamburg, Germany) to polarize microglia to an anti-inflammatory phenotype [ ].

Techniques: Expressing, Staining, Fluorescence, Microscopy, Marker, In Vitro, Quantitative RT-PCR

Activation characteristics of primary microglia. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to different experimental groups as marked by a horizontal bar. Characterization of the activated pro-inflammatory microglia phenotype by the expression of the inducible nitric oxide (NO) synthetase (iNOS) and release of NO after blockade of HCN and KCNQ/Kv7 channels with ZD7288 and XE-991 (30 μM each), respectively. Treatment with LPS (10 ng/ml) served as a positive control. a . iNOS expression was measured on RNA level by RT-qPCR (H(3) = 11.608, p < 0.009) and on b protein level by immunocytochemistry (H(3) = 17.665, p = 0.001). c Release of NO was measured by Griess assay (μmol/l; H(3) = 15.784, p = 0.001). Characterization of the anti-inflammatory phenotype of activated microglia by expression of CD206, release of insulin-like growth factor 1 (IGF1), and change of phagocytic capacity, after pharmacological block of HCN and KCNQ/Kv7 channels by 30 μM ZD7288 and XE-991, respectively. Treatment with IL4 (25 ng/ml) served as a positive control. d Regulation of CD206 expression on RNA level was measured by RT-qPCR (F (3, 20) = 13.981, p < 0.0001, ω = 0.62). e IGF1 release was measured by ELISA (pg/ml, H(3) = 15.805, p = 0.001). f Zymosan engulfment showed phagocytic activity of microglia (H(3) = 11.653, p < 0.009). Data were blank-corrected and normalized to control. g Representative immunocytochemical stainings for the microglia marker Iba1 (red), co-stained for iNOS (green), and Hoechst as a nuclear counterstain (blue); scale bars = 50 μm

Journal: Journal of Neuroinflammation

Article Title: The impact of hyperpolarization-activated cyclic nucleotide-gated (HCN) and voltage-gated potassium KCNQ/Kv7 channels on primary microglia function

doi: 10.1186/s12974-020-01779-4

Figure Lengend Snippet: Activation characteristics of primary microglia. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to different experimental groups as marked by a horizontal bar. Characterization of the activated pro-inflammatory microglia phenotype by the expression of the inducible nitric oxide (NO) synthetase (iNOS) and release of NO after blockade of HCN and KCNQ/Kv7 channels with ZD7288 and XE-991 (30 μM each), respectively. Treatment with LPS (10 ng/ml) served as a positive control. a . iNOS expression was measured on RNA level by RT-qPCR (H(3) = 11.608, p < 0.009) and on b protein level by immunocytochemistry (H(3) = 17.665, p = 0.001). c Release of NO was measured by Griess assay (μmol/l; H(3) = 15.784, p = 0.001). Characterization of the anti-inflammatory phenotype of activated microglia by expression of CD206, release of insulin-like growth factor 1 (IGF1), and change of phagocytic capacity, after pharmacological block of HCN and KCNQ/Kv7 channels by 30 μM ZD7288 and XE-991, respectively. Treatment with IL4 (25 ng/ml) served as a positive control. d Regulation of CD206 expression on RNA level was measured by RT-qPCR (F (3, 20) = 13.981, p < 0.0001, ω = 0.62). e IGF1 release was measured by ELISA (pg/ml, H(3) = 15.805, p = 0.001). f Zymosan engulfment showed phagocytic activity of microglia (H(3) = 11.653, p < 0.009). Data were blank-corrected and normalized to control. g Representative immunocytochemical stainings for the microglia marker Iba1 (red), co-stained for iNOS (green), and Hoechst as a nuclear counterstain (blue); scale bars = 50 μm

Techniques: Activation Assay, Expressing, Positive Control, Quantitative RT-PCR, Immunocytochemistry, Griess Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Marker, Staining

Impact of functional HCN and KCNQ/Kv7 channels on the activation capacity of primary microglia. * p < 0.05; ** p < 0.01; *** p < 0.001. Stimulation of microglia with LPS (10 ng/ml) with simultaneous blockade of the HCN or the KCNQ/Kv7 channel and resulting expression of pro-inflammatory markers. The HCN channel was either blocked pharmacologically with ZD7288 (30 μM) or by transfection of silencer® small interfering (si)RNA targeting HCN2-mRNA (siHCN2-RNA). XE-991 (30 μM) was used to block KCNQ/Kv7. a Inducible nitric oxide (NO) synthase (iNOS) expression was measured on RNA level by RT-qPCR (H(3) = 17.142, p = 0.001) and on b protein level by immunocytochemistry (H(3) = 19.626, p < 0.0001). c NO release was measured by Griess assay (μmol/l; H(3) = 23.102, p < 0.0001). Data were normalized to LPS-only stimulation. Characterization of the anti-inflammatory microglia phenotype by the expression of CD206, release of insulin-like growth factor 1 (IGF1), and measurement of the phagocytic activity, after treatment with IL4 (50 ng/ml) and simultaneous blockade of the HCN and KCNQ/Kv7 channel as described above. d Regulation of CD206 expression was measured on RNA level by RT-qPCR (H(3) = 12.755, p = 0.005). e Release of IGF1 was measured by ELISA (pg/ml; H(3) = 16.611, p = 0.001). f Zymosan engulfment indicated phagocytotic activity of microglia (H(2) = 7.501, p = 0.024). Data were normalized to IL4-only stimulation. g Representative images of the immunocytochemical staining for the microglia marker Iba1 (red), co-stained for iNOS (green), and Hoechst as a nuclear counterstain (blue); scale bars = 50 μm

Journal: Journal of Neuroinflammation

Article Title: The impact of hyperpolarization-activated cyclic nucleotide-gated (HCN) and voltage-gated potassium KCNQ/Kv7 channels on primary microglia function

doi: 10.1186/s12974-020-01779-4

Figure Lengend Snippet: Impact of functional HCN and KCNQ/Kv7 channels on the activation capacity of primary microglia. * p < 0.05; ** p < 0.01; *** p < 0.001. Stimulation of microglia with LPS (10 ng/ml) with simultaneous blockade of the HCN or the KCNQ/Kv7 channel and resulting expression of pro-inflammatory markers. The HCN channel was either blocked pharmacologically with ZD7288 (30 μM) or by transfection of silencer® small interfering (si)RNA targeting HCN2-mRNA (siHCN2-RNA). XE-991 (30 μM) was used to block KCNQ/Kv7. a Inducible nitric oxide (NO) synthase (iNOS) expression was measured on RNA level by RT-qPCR (H(3) = 17.142, p = 0.001) and on b protein level by immunocytochemistry (H(3) = 19.626, p < 0.0001). c NO release was measured by Griess assay (μmol/l; H(3) = 23.102, p < 0.0001). Data were normalized to LPS-only stimulation. Characterization of the anti-inflammatory microglia phenotype by the expression of CD206, release of insulin-like growth factor 1 (IGF1), and measurement of the phagocytic activity, after treatment with IL4 (50 ng/ml) and simultaneous blockade of the HCN and KCNQ/Kv7 channel as described above. d Regulation of CD206 expression was measured on RNA level by RT-qPCR (H(3) = 12.755, p = 0.005). e Release of IGF1 was measured by ELISA (pg/ml; H(3) = 16.611, p = 0.001). f Zymosan engulfment indicated phagocytotic activity of microglia (H(2) = 7.501, p = 0.024). Data were normalized to IL4-only stimulation. g Representative images of the immunocytochemical staining for the microglia marker Iba1 (red), co-stained for iNOS (green), and Hoechst as a nuclear counterstain (blue); scale bars = 50 μm

Techniques: Functional Assay, Activation Assay, Expressing, Transfection, Blocking Assay, Quantitative RT-PCR, Immunocytochemistry, Griess Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Staining, Marker

Journal: Brain, behavior, and immunity

Article Title: Immunization with Mycobacterium vaccae induces an anti-inflammatory milieu in the CNS: attenuation of stress-induced microglial priming, alarmins and anxiety-like behavior

doi: 10.1016/j.bbi.2018.05.020

Figure Lengend Snippet: Primer Specifications.

Article Snippet: Recombinant rat IL4 (rIL4) treatment Vehicle (sterile 1x PBS + 0.1% BSA) or rIL4 (100 ng dissolved in vehicle; R & D Systems, cat. no. 504-RL) was injected intra-cisterna magna (i.c.m.

Techniques: Sequencing, Activation Assay

Animals received injections of either vehicle or rIL4 (100 ng, i.c.m.). IL4-sensitive target genes were measured in hippocampus at 2 h (N = 6–8 per group) or 24 h post-injection (N = 4 per group). Data are presented as the mean + s.e.m. Significant M. vaccae effects compared to vehicle at each timepoint post-injection, * p < 0.05, ** p < 0.01.

Journal: Brain, behavior, and immunity

Article Title: Immunization with Mycobacterium vaccae induces an anti-inflammatory milieu in the CNS: attenuation of stress-induced microglial priming, alarmins and anxiety-like behavior

doi: 10.1016/j.bbi.2018.05.020

Figure Lengend Snippet: Animals received injections of either vehicle or rIL4 (100 ng, i.c.m.). IL4-sensitive target genes were measured in hippocampus at 2 h (N = 6–8 per group) or 24 h post-injection (N = 4 per group). Data are presented as the mean + s.e.m. Significant M. vaccae effects compared to vehicle at each timepoint post-injection, * p < 0.05, ** p < 0.01.

Techniques: Injection

Animals received 3 injections of either vehicle or M. vaccae (0.1 mg, s.c.). Eight d after the third injection, (A) IL4 mRNA expression and (B) IL4 protein levels were measured in dorsal, intermediate, and ventral hippocampus. Data are presented as the mean + s.e.m. N = 8 animals per experimental group. Significant main effect of M. vaccae compared to vehicle, * p < 0.05.

Journal: Brain, behavior, and immunity

Article Title: Immunization with Mycobacterium vaccae induces an anti-inflammatory milieu in the CNS: attenuation of stress-induced microglial priming, alarmins and anxiety-like behavior

doi: 10.1016/j.bbi.2018.05.020

Figure Lengend Snippet: Animals received 3 injections of either vehicle or M. vaccae (0.1 mg, s.c.). Eight d after the third injection, (A) IL4 mRNA expression and (B) IL4 protein levels were measured in dorsal, intermediate, and ventral hippocampus. Data are presented as the mean + s.e.m. N = 8 animals per experimental group. Significant main effect of M. vaccae compared to vehicle, * p < 0.05.

Techniques: Injection, Expressing

Schematic Illustration of Preparation of IL4-Loaded NHG-MSa

Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: Schematic Illustration of Preparation of IL4-Loaded NHG-MSa

Article Snippet: Recombinant rat IL4 (R&D system) was reconstituted at a concentration of 4 μ g/mL in sterile Dulbecco’s phosphate-buffered saline (DPBS) (Gibco) containing 0.1% bovine serum albumin (BSA, Sigma-Aldrich).

Techniques:

Characterizations of IL4-loaded NHG-MS and NG-MS and their release profiles. (A–C) Stacked confocal images of IL4-loaded NHG-MS. (D–F) Cross-sectional confocal images at a higher magnification, showing that IL4 (red) was evenly distributed in the NHG-MS (green). (G) Loading efficiency of IL4 in NHG-MS and NG-MS. (H) Release profiles of IL4 from NHG-MS and NG-MS. (I) Total amount of IL4 (released and unreleased) from the NHG-MS and NG-MS detected via an ELISA (**P < 0.01).

Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: Characterizations of IL4-loaded NHG-MS and NG-MS and their release profiles. (A–C) Stacked confocal images of IL4-loaded NHG-MS. (D–F) Cross-sectional confocal images at a higher magnification, showing that IL4 (red) was evenly distributed in the NHG-MS (green). (G) Loading efficiency of IL4 in NHG-MS and NG-MS. (H) Release profiles of IL4 from NHG-MS and NG-MS. (I) Total amount of IL4 (released and unreleased) from the NHG-MS and NG-MS detected via an ELISA (**P < 0.01).

Techniques: Enzyme-linked Immunosorbent Assay

In vitro bioactivity assay of the IL4 released from NHG-MS. Bone marrow-derived macrophages (BMDMs) stimulated by LPS and IFN-γ for 24 h were polarized into M1 proinflammatory macrophages and then exposed to IL4 released from NHG-MS at 1 and 14 days for 24 h. The LPS + IFN-γ-only and the LPS + IFN-γ + IL4 groups were used as negative and positive controls, respectively. (A, B) Immunofluorescence images and quantitative analysis of CD206+ (green, an M2 macrophage marker) and CD68+ (red, pan-macrophage marker) BMDMs. Cells treated with the LPS + IFN-γ-only group exhibited a rounded morphology, and CD206+ cells were barely detected in this negative control group, indicating the proinflammatory M1 phenotype. By contrast, addition of IL4 released from NHG-MS at 1 and 14 days switched the cells into typical elongated spindle shapes, and approximately 70% BMDMs were CD206+CD68+ M2 phenotype, which was similar to the IL4 positive control group. (C, D) Quantitative analysis of proinflammatory cytokines in the supernatant showed the significant reduction of both TNF-α and IL6 in three groups with additional IL4 treatment (**P < 0.01).

Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: In vitro bioactivity assay of the IL4 released from NHG-MS. Bone marrow-derived macrophages (BMDMs) stimulated by LPS and IFN-γ for 24 h were polarized into M1 proinflammatory macrophages and then exposed to IL4 released from NHG-MS at 1 and 14 days for 24 h. The LPS + IFN-γ-only and the LPS + IFN-γ + IL4 groups were used as negative and positive controls, respectively. (A, B) Immunofluorescence images and quantitative analysis of CD206+ (green, an M2 macrophage marker) and CD68+ (red, pan-macrophage marker) BMDMs. Cells treated with the LPS + IFN-γ-only group exhibited a rounded morphology, and CD206+ cells were barely detected in this negative control group, indicating the proinflammatory M1 phenotype. By contrast, addition of IL4 released from NHG-MS at 1 and 14 days switched the cells into typical elongated spindle shapes, and approximately 70% BMDMs were CD206+CD68+ M2 phenotype, which was similar to the IL4 positive control group. (C, D) Quantitative analysis of proinflammatory cytokines in the supernatant showed the significant reduction of both TNF-α and IL6 in three groups with additional IL4 treatment (**P < 0.01).

Techniques: In Vitro, Derivative Assay, Immunofluorescence, Marker, Negative Control, Positive Control

Macrophage phenotype in the defect area 7 days after surgical operation: iNOS (green, an M1 macrophage marker); CD206 (green, an M2 macrophage marker); and CD68 (red, pan-macrophage marker). (A) Immunofluorescence images showed that iNOS was extensively expressed in the DM and DM + NHG-MS groups. In comparison, the expression of iNOS in the IL4-loaded NHG-MS DM group was much less, which is similar to the normal control group, indicating fewer M1 macrophages in the normal control and IL4-loaded NHG-MS DM groups than the other two DM groups. (B) Immunofluorescence images showed that the expression of CD206 in the IL4-loaded NHG-MS DM group was similar to that in the normal control group and much higher than that in the other two DM groups. (C–F) Quantitative analysis showed that the expression of CD68 was significantly higher in the three DM groups than that in the normal control group (C), indicating that more macrophages infiltrated into the defect site of diabetic rats. Fewer M1 macrophages and more M2 macrophages in both the normal control and IL4-loaded NHG-MS DM groups (D, E) resulted in significantly higher M2/M1 ratios than in the other two DM groups (F) (**P < 0.01).

Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: Macrophage phenotype in the defect area 7 days after surgical operation: iNOS (green, an M1 macrophage marker); CD206 (green, an M2 macrophage marker); and CD68 (red, pan-macrophage marker). (A) Immunofluorescence images showed that iNOS was extensively expressed in the DM and DM + NHG-MS groups. In comparison, the expression of iNOS in the IL4-loaded NHG-MS DM group was much less, which is similar to the normal control group, indicating fewer M1 macrophages in the normal control and IL4-loaded NHG-MS DM groups than the other two DM groups. (B) Immunofluorescence images showed that the expression of CD206 in the IL4-loaded NHG-MS DM group was similar to that in the normal control group and much higher than that in the other two DM groups. (C–F) Quantitative analysis showed that the expression of CD68 was significantly higher in the three DM groups than that in the normal control group (C), indicating that more macrophages infiltrated into the defect site of diabetic rats. Fewer M1 macrophages and more M2 macrophages in both the normal control and IL4-loaded NHG-MS DM groups (D, E) resulted in significantly higher M2/M1 ratios than in the other two DM groups (F) (**P < 0.01).

Techniques: Marker, Immunofluorescence, Comparison, Expressing, Control

Expression of proinflammatory cytokines TNF-α in the defect area 7 days after the surgical operation. (A–D) Immunohistochemical images showed stronger expression of TNF-α in DM and DM + NHG-MS groups than in normal control and IL4-loaded NHG-MS DM groups. (E, F) Quantitative analysis of TNF-α positive area ratio and mean integral optical density (IOD) (**P < 0.01).

Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: Expression of proinflammatory cytokines TNF-α in the defect area 7 days after the surgical operation. (A–D) Immunohistochemical images showed stronger expression of TNF-α in DM and DM + NHG-MS groups than in normal control and IL4-loaded NHG-MS DM groups. (E, F) Quantitative analysis of TNF-α positive area ratio and mean integral optical density (IOD) (**P < 0.01).

Techniques: Expressing, Immunohistochemical staining, Control

Expression of Osterix and Runx2 in the defect area 14 days after the surgical operation. (A, B) Immunohistochemical images showed significantly more Osterix+ and Runx2+ osteoprogenitor cells in normal control and IL4-loaded NHG-MS DM groups than in the other two DM groups. (C–F) Quantitative analysis of the number of Osterix+ and Runx2+ cells and mean integral optical density (IOD) (*P < 0.05, **P < 0.01).

Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: Expression of Osterix and Runx2 in the defect area 14 days after the surgical operation. (A, B) Immunohistochemical images showed significantly more Osterix+ and Runx2+ osteoprogenitor cells in normal control and IL4-loaded NHG-MS DM groups than in the other two DM groups. (C–F) Quantitative analysis of the number of Osterix+ and Runx2+ cells and mean integral optical density (IOD) (*P < 0.05, **P < 0.01).

Techniques: Expressing, Immunohistochemical staining, Control

ALP staining of the defect area 14 days after the surgical operation. (A–D) Normal control and IL4-loaded NHG-MS DM groups exhibited significantly more extensive and robust ALP expression than in the other two DM groups. (E, F) Quantitative analysis of ALP-positive area ratio and mean integral optical density (IOD) (*P < 0.05, **P < 0.01).

Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: ALP staining of the defect area 14 days after the surgical operation. (A–D) Normal control and IL4-loaded NHG-MS DM groups exhibited significantly more extensive and robust ALP expression than in the other two DM groups. (E, F) Quantitative analysis of ALP-positive area ratio and mean integral optical density (IOD) (*P < 0.05, **P < 0.01).

Techniques: Staining, Control, Expressing

μ-CT three-dimensional reconstruction images of the rat mandible and new regenerated bone 4 weeks after surgery. (A–H) The defect area was entirely occupied by new regenerated bone in both the normal control group and the IL4-loaded NHG-MS DM group. In contrast, only half of the defect region was filled with new bone in the DM and DM + NHG-MS groups. (I) Ratio of bone volume to total volume (BV/TV) in all four groups (**P < 0.01).

Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: μ-CT three-dimensional reconstruction images of the rat mandible and new regenerated bone 4 weeks after surgery. (A–H) The defect area was entirely occupied by new regenerated bone in both the normal control group and the IL4-loaded NHG-MS DM group. In contrast, only half of the defect region was filled with new bone in the DM and DM + NHG-MS groups. (I) Ratio of bone volume to total volume (BV/TV) in all four groups (**P < 0.01).

Techniques: Control

HE staining of the defect area 4 weeks after the surgical operation. (A–D) Newly formed bone filled the entire defect area in the normal control and the IL4-loaded NHG-MS DM group. In comparison, new bone formation was observed on the outer boundary of the defect area in the DM and DM + NHG-MS groups. The yellow arrows indicate newly formed bone, and the blue arrows indicate microspheres.

Journal: ACS applied materials & interfaces

Article Title: Immunomodulatory ECM-like Microspheres for Accelerated Bone Regeneration in Diabetes Mellitus

doi: 10.1021/acsami.7b18458

Figure Lengend Snippet: HE staining of the defect area 4 weeks after the surgical operation. (A–D) Newly formed bone filled the entire defect area in the normal control and the IL4-loaded NHG-MS DM group. In comparison, new bone formation was observed on the outer boundary of the defect area in the DM and DM + NHG-MS groups. The yellow arrows indicate newly formed bone, and the blue arrows indicate microspheres.

Techniques: Staining, Control, Comparison

Figure 1. NanoString analysis of expression changes for selected genes in response to IL4- and IL10-treatment. Treating rat primary microglia for 6 hr with 20 ng/ml IL4 or 20 ng/ml IL10 resulted in gene expression changes that were specific for IL4 (A), specific for IL10 (B), or common to both treatments (C). mRNA expression was normalized to the housekeeping gene, HPRT1, and shown as mean 6 SEM with the number

Journal: PloS one

Article Title: Expression and contributions of TRPM7 and KCa2.3/SK3 channels to the increased migration and invasion of microglia in anti-inflammatory activation states.

doi: 10.1371/journal.pone.0106087

Figure Lengend Snippet: Figure 1. NanoString analysis of expression changes for selected genes in response to IL4- and IL10-treatment. Treating rat primary microglia for 6 hr with 20 ng/ml IL4 or 20 ng/ml IL10 resulted in gene expression changes that were specific for IL4 (A), specific for IL10 (B), or common to both treatments (C). mRNA expression was normalized to the housekeeping gene, HPRT1, and shown as mean 6 SEM with the number

Article Snippet: Chemicals Primary microglia and MLS-9 cells were stimulated with 20 ng/ml rat recombinant IL4 or 20 ng/ml rat recombinant IL10 (both from R&D Systems Inc., Minneapolis, MN).

Techniques: Expressing, Gene Expression

Figure 2. Effect of microglial activation state and selected channel inhibitors on podosome (podonut) expression. A. Representative fluorescence micrographs show podonut expression in unstimulated primary rat microglia or 24 hr after treatment with LPS (10 ng/ml), IL4 (20 ng/ ml) or IL10 (20 ng/ml). The arrows indicate examples of a single, large donut-shaped ring of podosomes, which we call a ‘podonut’ in the lamellum of unipolar microglia. Cells were stained for filamentous (F-) actin with phalloidin (green) to quantify the proportion of cells with a podonut, and with the nuclear marker, DAPI (blue) to quantify total cell numbers. Scale bar, 50 mm. B. Podosome (podonut) expression is affected by the microglial activation state. The proportion of microglial cells bearing a podonut in the lamellum (sum of 3 randomly selected fields of view at 106 magnification) was normalized to unstimulated cells. C–E. Microglia were unstimulated or stimulated for 24 hr with 20 ng/ml IL4 or IL10, and then exposed for 6 hr to control medium or the KCa2.3 inhibitors, 5 nM tamapin or 7 mM NS8593, which also inhibits TRPM7 channels (see Results and Figs. 6, 7). All graphical data are shown as mean 6 SEM with sample size (# of individual cultures) indicated on each bar. A one-way ANOVA with Tukey’s post-hoc analysis was used to determine significant differences. *p,0.05; ***p,0.001. doi:10.1371/journal.pone.0106087.g002

Journal: PloS one

Article Title: Expression and contributions of TRPM7 and KCa2.3/SK3 channels to the increased migration and invasion of microglia in anti-inflammatory activation states.

doi: 10.1371/journal.pone.0106087

Figure Lengend Snippet: Figure 2. Effect of microglial activation state and selected channel inhibitors on podosome (podonut) expression. A. Representative fluorescence micrographs show podonut expression in unstimulated primary rat microglia or 24 hr after treatment with LPS (10 ng/ml), IL4 (20 ng/ ml) or IL10 (20 ng/ml). The arrows indicate examples of a single, large donut-shaped ring of podosomes, which we call a ‘podonut’ in the lamellum of unipolar microglia. Cells were stained for filamentous (F-) actin with phalloidin (green) to quantify the proportion of cells with a podonut, and with the nuclear marker, DAPI (blue) to quantify total cell numbers. Scale bar, 50 mm. B. Podosome (podonut) expression is affected by the microglial activation state. The proportion of microglial cells bearing a podonut in the lamellum (sum of 3 randomly selected fields of view at 106 magnification) was normalized to unstimulated cells. C–E. Microglia were unstimulated or stimulated for 24 hr with 20 ng/ml IL4 or IL10, and then exposed for 6 hr to control medium or the KCa2.3 inhibitors, 5 nM tamapin or 7 mM NS8593, which also inhibits TRPM7 channels (see Results and Figs. 6, 7). All graphical data are shown as mean 6 SEM with sample size (# of individual cultures) indicated on each bar. A one-way ANOVA with Tukey’s post-hoc analysis was used to determine significant differences. *p,0.05; ***p,0.001. doi:10.1371/journal.pone.0106087.g002

Article Snippet: Chemicals Primary microglia and MLS-9 cells were stimulated with 20 ng/ml rat recombinant IL4 or 20 ng/ml rat recombinant IL10 (both from R&D Systems Inc., Minneapolis, MN).

Techniques: Activation Assay, Expressing, Fluorescence, Staining, Marker, Control

Figure 4. Migration of MLS-9 microglial cells is increased by IL4 and IL10, and involves TRPM7, not KCa2.3. A. Representative fluorescence micrographs show MLS-9 cells (rat microglial cell line), unstimulated or 24 hr after stimulation with LPS (100 ng/ml), IL4 (20 ng/ml) or IL10 (20 ng/ml). Cells were stained for F-actin with phalloidin (green) and the nuclear marker, DAPI (blue). Most cells were bipolar with membrane ruffling at one end (examples shown by arrows). Scale bar, 50 mm. B. MLS-9 microglial cells in the upper well of Transwells were unstimulated, or stimulated (24 hr) with 20 or 100 ng/ml IL4, or with 20 or 100 ng/ml IL10. The number of cells that migrated to the underside of the filter was then counted and normalized to control (unstimulated) cells. The dashed line in both graphs indicates the level in control (unstimulated) cells. C. Cells were stimulated as in panel B, with or without a channel inhibitor: 100 nM apamin, 5 nM tamapin, or 7 mM NS8593. For each stimulus, the number of cells that had migrated was normalized to the level without a channel inhibitor. Data are expressed as mean 6 SEM with the number of individual cultures indicated on each bar. In panel B, a two-way ANOVA with Bonferroni post-hoc analysis was used to determine significant differences between stimulation (*) and dose ({). In panel C, A one-way ANOVA with Tukey’s post-hoc test was used to determine treatment effects. One symbol, p,0.05; two symbols, p,0.01; three symbols, p,0.001. doi:10.1371/journal.pone.0106087.g004

Journal: PloS one

Article Title: Expression and contributions of TRPM7 and KCa2.3/SK3 channels to the increased migration and invasion of microglia in anti-inflammatory activation states.

doi: 10.1371/journal.pone.0106087

Figure Lengend Snippet: Figure 4. Migration of MLS-9 microglial cells is increased by IL4 and IL10, and involves TRPM7, not KCa2.3. A. Representative fluorescence micrographs show MLS-9 cells (rat microglial cell line), unstimulated or 24 hr after stimulation with LPS (100 ng/ml), IL4 (20 ng/ml) or IL10 (20 ng/ml). Cells were stained for F-actin with phalloidin (green) and the nuclear marker, DAPI (blue). Most cells were bipolar with membrane ruffling at one end (examples shown by arrows). Scale bar, 50 mm. B. MLS-9 microglial cells in the upper well of Transwells were unstimulated, or stimulated (24 hr) with 20 or 100 ng/ml IL4, or with 20 or 100 ng/ml IL10. The number of cells that migrated to the underside of the filter was then counted and normalized to control (unstimulated) cells. The dashed line in both graphs indicates the level in control (unstimulated) cells. C. Cells were stimulated as in panel B, with or without a channel inhibitor: 100 nM apamin, 5 nM tamapin, or 7 mM NS8593. For each stimulus, the number of cells that had migrated was normalized to the level without a channel inhibitor. Data are expressed as mean 6 SEM with the number of individual cultures indicated on each bar. In panel B, a two-way ANOVA with Bonferroni post-hoc analysis was used to determine significant differences between stimulation (*) and dose ({). In panel C, A one-way ANOVA with Tukey’s post-hoc test was used to determine treatment effects. One symbol, p,0.05; two symbols, p,0.01; three symbols, p,0.001. doi:10.1371/journal.pone.0106087.g004

Article Snippet: Chemicals Primary microglia and MLS-9 cells were stimulated with 20 ng/ml rat recombinant IL4 or 20 ng/ml rat recombinant IL10 (both from R&D Systems Inc., Minneapolis, MN).

Techniques: Migration, Fluorescence, Staining, Marker, Membrane, Control

Figure 5. KCNN3 expression and KCa2.3 current inhibition by NS8593 in microglia in differing activation states. A. Expression of KCNN3 mRNA was quantified using NanoString nCounter analysis in unstimulated primary microglia, and at 6 hr (solid bars) and 24 hr (striped bars) after treatment with 20 ng/ml IL4- or IL10-treatment. B–D. NS8593 inhibits the KCa2.3 current, while AA-861 has no effect. KCa2.3 currents were

Journal: PloS one

Article Title: Expression and contributions of TRPM7 and KCa2.3/SK3 channels to the increased migration and invasion of microglia in anti-inflammatory activation states.

doi: 10.1371/journal.pone.0106087

Figure Lengend Snippet: Figure 5. KCNN3 expression and KCa2.3 current inhibition by NS8593 in microglia in differing activation states. A. Expression of KCNN3 mRNA was quantified using NanoString nCounter analysis in unstimulated primary microglia, and at 6 hr (solid bars) and 24 hr (striped bars) after treatment with 20 ng/ml IL4- or IL10-treatment. B–D. NS8593 inhibits the KCa2.3 current, while AA-861 has no effect. KCa2.3 currents were

Article Snippet: Chemicals Primary microglia and MLS-9 cells were stimulated with 20 ng/ml rat recombinant IL4 or 20 ng/ml rat recombinant IL10 (both from R&D Systems Inc., Minneapolis, MN).

Techniques: Expressing, Inhibition, Activation Assay

Figure 8. Effects of IL4- and IL10-treatment on TRPM7 expression, current and block by NS8593 in primary rat microglia. A. TRPM7 mRNA expression was measured using quantitative real-time RT-PCR at 6 hr (solid bars) and 24 hr (striped bars) in unstimulated rat microglia or after treatment with 20 ng/ml IL4 or 20 ng/ml IL10. Values are expressed as mean expression (normalized to HPRT1) 6 SEM, with the number of individual cultures indicated on each bar. *p,0.05 indicates the difference from time-matched control (unstimulated) cells, and was determined using a 2-way ANOVA followed by Bonferroni post-hoc test. B–E. The TRPM7 current and block by NS8593 were not affected by 24 hr treatment with 20 ng/ml IL4 or IL10. Whole-cell recordings were performed on primary rat microglia using Mg2+-free pipette solution. B, C. Representative currents (upper panels) in response to 120 ms-long ramps from 2100 to +115 mV, from a holding potential of 210 mV. Time course of the current (lower panels) measured at +115 mV. D, E. Summary of TRPM7 current amplitudes measured at +115 mV (D) and percent inhibition of TRPM7 currents by 7 mM NS8593 (E). Graphical data are presented as mean 6 SEM for the number of cells indicated on each bar, and were compared using a one-way ANOVA with Tukey’s post-hoc test. doi:10.1371/journal.pone.0106087.g008

Journal: PloS one

Article Title: Expression and contributions of TRPM7 and KCa2.3/SK3 channels to the increased migration and invasion of microglia in anti-inflammatory activation states.

doi: 10.1371/journal.pone.0106087

Figure Lengend Snippet: Figure 8. Effects of IL4- and IL10-treatment on TRPM7 expression, current and block by NS8593 in primary rat microglia. A. TRPM7 mRNA expression was measured using quantitative real-time RT-PCR at 6 hr (solid bars) and 24 hr (striped bars) in unstimulated rat microglia or after treatment with 20 ng/ml IL4 or 20 ng/ml IL10. Values are expressed as mean expression (normalized to HPRT1) 6 SEM, with the number of individual cultures indicated on each bar. *p,0.05 indicates the difference from time-matched control (unstimulated) cells, and was determined using a 2-way ANOVA followed by Bonferroni post-hoc test. B–E. The TRPM7 current and block by NS8593 were not affected by 24 hr treatment with 20 ng/ml IL4 or IL10. Whole-cell recordings were performed on primary rat microglia using Mg2+-free pipette solution. B, C. Representative currents (upper panels) in response to 120 ms-long ramps from 2100 to +115 mV, from a holding potential of 210 mV. Time course of the current (lower panels) measured at +115 mV. D, E. Summary of TRPM7 current amplitudes measured at +115 mV (D) and percent inhibition of TRPM7 currents by 7 mM NS8593 (E). Graphical data are presented as mean 6 SEM for the number of cells indicated on each bar, and were compared using a one-way ANOVA with Tukey’s post-hoc test. doi:10.1371/journal.pone.0106087.g008

Article Snippet: Chemicals Primary microglia and MLS-9 cells were stimulated with 20 ng/ml rat recombinant IL4 or 20 ng/ml rat recombinant IL10 (both from R&D Systems Inc., Minneapolis, MN).

Techniques: Expressing, Blocking Assay, Quantitative RT-PCR, Control, Transferring, Inhibition

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