Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3a in B cells is generated extracellularly by alternative pathway C3-convertases. Western blot results investigating the origin of C3a in Raji B cells. Cells were incubated with distinct sources of C3 (10% NHS, 100 μg/ml C3, 100 μg/ml C3met) in DGVB++, Mg-EGTA, or EDTA-GVB buffer for 1 h at 37 °C. To analyze complement pathway dependent generation of C3a, sera depleted in C1q (classical pathway), MBL (lectin pathway) or Factor B (alternative pathway) were used. To distinguish intracellular or C3-convertase dependent processing of C3, C3, and C3met were added alone (intracellular cleavage) or in the presence of C3-depleted serum (C3 convertase supplementation). C3 processing and C3a generation were analyzed by Western blot with the goat polyclonal anti-C3 (Quidel) and rabbit anti-C3a (Complement Technologies) antibodies under reducing conditions. Result shown is one representative out of three independent experiments.

Article Snippet: C3a was detected with the polyclonal rabbit anti-C3a antibody ( ) from Complement Technologies (#A218, Western blot) or with the monoclonal mouse anti-C3a/C3adesArg Ab from Hycult (#HM2074, gel shift assays and DNA ELISA).

Techniques: Generated, Western Blot, Incubation

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3 and C3a enter the nucleus after uptake. (A,B) Western blot results showing presence of C3 and C3a in nuclear compartments of Raji cells. The human B cell line, Raji was incubated with NHS, C3 or C3met as a source of C3 either in EDTA-GVB (A) or in Mg-EGTA (B) buffer for 1 h at 37°C. After lysis, cytoplasmic, soluble nuclear, and chromatin-associated nuclear fractions were separated and analyzed by Western blot with the goat polyclonal anti-C3 antibody under non reducing conditions (A) or with the rabbit polyclonal antibody against C3a under reducing condition (B) . The purity of distinct cellular fractions was verified using antibodies against B-actin (cytoplasmic marker), lamin B1 (nuclear marker) and histone H2B (chromatin-associated nuclear marker). Results shown are one representative experiment out of three (A) or four (B) independent analyzes. (C) Representative confocal images showing that AlexaFluor 488 labeled C3 enters the nucleus. 2 × 10 6 Raji cells were incubated with 100 μg/ml C3-AlexaFluor 488 for 30 min at 37°C, fixed and counterstained with DAPI using mounting medium. Representative images are shown from two independent experiments investigating at least 50 cells/analysis.

Article Snippet: C3a was detected with the polyclonal rabbit anti-C3a antibody ( ) from Complement Technologies (#A218, Western blot) or with the monoclonal mouse anti-C3a/C3adesArg Ab from Hycult (#HM2074, gel shift assays and DNA ELISA).

Techniques: Western Blot, Incubation, Lysis, Marker, Labeling

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: C3 and C3a bind to distinct types of DNA. (A–C) Gel shift assays presenting interaction between C3(a) and genomic DNA isolated from Raji cells. Isolated genomic DNA was incubated either with pre-titrated concentrations of C3 (A) or C3a (B) or C3b (C) and separated by agarose gel electrophoresis. Formation of high molecular weight DNA-protein complexes is indicated by slower DNA migration (DNA shift) compared to migration of the free nucleic acid. The presence of C3 and its cleavage fragments in the pre-formed protein-DNA complexes was confirmed with anti-C3a and anti-C3d antibodies caused supershift. One representative experiment out of five performed is shown. (D) Gel shift assays showing that C3(a)—DNA interaction is not ionic in nature. C3 or C3a were incubated with genomic DNA of Raji cells in the presence of increasing NaCl concentration (ranging from 150 to 1,200 mM). Binding of C3 and C3a to DNA was observed at 8X higher salt concentration than the physiological 150 mM. (E) Interaction of C3 and C3a with DNA is not restricted to genomic DNA. Linearized pCEP4 vector was incubated with either C3 or C3a and DNA-protein complex formation was investigated by gel shift assay. Results illustrated are one representative out of three independent experiments. (F,G) ELISA results confirming interaction between C3(a) and DNA. ELISA microplate surfaces were coated either with DNA (F) or C3, C3a, C3b (G) . After incubation with distinct concentrations of C3 proteins (F) or Raji genomic DNA (G) , protein-DNA interactions were detected using anti-C3d, anti-C3a (anti-C3 ELISA, F ), or anti-dsDNA antibody (anti-DNA ELISA, G ). Results shown are mean absorbance values measured at 490 nm with SD of four independent experiments. (H) ELISA results showing presence of C3-DNA complexes in formaldehyde cross-linked chromatin fraction of Raji B cells. Cells were incubated in the presence or absence of C3, C3met, fixed with 1% formaldehyde to cross-link protein-DNA complexes and chromatin isolated using commercial kit. C3-DNA complexes were investigated by ELISA on anti-C3 or anti-DNA coated plates. Data are shown as mean absorbance values measured at 490 nm with SD of three independent experiments. Differences with p < 0.05 were considered statistically significant and compared to non-treated cells (two-way ANOVA with Dunnett's multiple comparison, ns p > 0.05, * p < 0.05, ** p < 0.01).

Article Snippet: C3a was detected with the polyclonal rabbit anti-C3a antibody ( ) from Complement Technologies (#A218, Western blot) or with the monoclonal mouse anti-C3a/C3adesArg Ab from Hycult (#HM2074, gel shift assays and DNA ELISA).

Techniques: Electrophoretic Mobility Shift Assay, Isolation, Incubation, Agarose Gel Electrophoresis, Molecular Weight, Migration, Concentration Assay, Binding Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Interaction of Serum-Derived and Internalized C3 With DNA in Human B Cells—A Potential Involvement in Regulation of Gene Transcription

doi: 10.3389/fimmu.2019.00493

Figure Lengend Snippet: Interaction of C3 and its cleavage fragments with histones. Recombinant histone core octamers (H2A/H2B/H3/H4), the linker H1 histone, or alpha-1-antitrypsin (negative control) were immobilized on microtiter plates and incubated with increasing concentrations of C3 (A) , C3b (B) , or C3a (C) . As negative control, BSA was used. Binding was detected either with monoclonal anti-C3d (A,B) or polyclonal anti-C3a (C) antibodies. Background signals obtained from BSA incubation were subtracted from the original values. Data are presented as mean absorbance values measured at 490 nm with SD of three independent experiments. Differences with p < 0.05 were considered statistically significant and compared to 0 μg/ml protein incubated surfaces (two-way ANOVA with Dunnett's multiple comparison, ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). Upper asterisks indicate statistical significance of H1-, lower asterisks of histone octamer coated surfaces compared to binding to A1AT. (D) gDNA of Raji cells was coated on microtiter plates and AlexaFluor 488-labeled H1 histone binding was monitored in the presence or absence of C3a. As negative control, BSA was used. Background signals obtained on A1AT coated surfaces were subtracted from the original values. Data are presented as relative fluorescent unit (RFU) measured at 525 nm with SD of two independent experiments. Differences with p < 0.05 were considered statistically significant and compared to no C3a treated samples (two-way ANOVA with Dunnett's multiple comparison, ns p > 0.05, * p < 0.05, ** p < 0.01). Upper and lower asterisks indicate statistical significance of H1-AlexaFluor 488 binding in the presence of BSA or C3a (respectively) compared to binding of H1-AlexaFluor 488 alone.

Article Snippet: C3a was detected with the polyclonal rabbit anti-C3a antibody ( ) from Complement Technologies (#A218, Western blot) or with the monoclonal mouse anti-C3a/C3adesArg Ab from Hycult (#HM2074, gel shift assays and DNA ELISA).

Techniques: Recombinant, Negative Control, Incubation, Binding Assay, Labeling

Journal: Clinical and experimental immunology

Article Title: Targeting C3a/C5a receptors inhibits human mesangial cell proliferation and alleviates immunoglobulin A nephropathy in mice.

doi: 10.1111/cei.12961

Figure Lengend Snippet: Fig. 1. Cell proliferation and cytokine mRNA and protein expression in cultured human mesangial cells (HMCs). The HMCs were treated with blank medium or medium containing 100 lg/ml immunoglobulin (Ig)A, 100 lg/ml IgA 1 100 lM C3aR antagonist (C3aRA) or 100 lg/ml IgA 1 100 lM C5aR antagonist (C5aRA) for 48 h. The data are expressed as the mean 6 standard deviation (s.d.). (a) HMC proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (n 5 6 for each group). (b) Interleukin (IL)-6 (left row) and monocyte chemotactic protein 1 (MCP-1) (right row) gene expression levels relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined by reverse transcription–quantitative polymerase chain reaction (RT–qPCR). (c) Western blot analysis of IL-6, MCP-1 and GAPDH protein expression. (d) Quantitative analysis for Western blot of IL-6 and MCP-1 protein expression. *P < 005, **P < 001, ***P < 0001 versus negative control.

Article Snippet: The membranes were blocked with 5% skimmed milk solution in 0 1% Tris-buffered saline Tween-20 (Dingguo Bioscience) for 1 h. The membranes were incubated subsequently with either mouse antihuman C3aR monoclonal antibodies (1 : 500 dilution; AbD Serotec, Oxford, UK), mouse anti-human C5aR monoclonal antibodies (1 : 500 dilution; AbD Serotec), rabbit anti-human interleukin (IL)-6 monoclonal antibodies (1 : 800 dilution; Abcam, Cambridge, UK), rabbit antihuman MCP-1 polyclonal antibodies (1:1000 dilution, Abcam) or rabbit anti-human GAPDH polyclonal antibodies (1 : 800 dilution; Good Here Bioscience, Hangzhou, China) overnight at 4 C with gentle shaking.

Techniques: Expressing, Cell Culture, Standard Deviation, MTT Assay, Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Negative Control

Journal: Clinical and experimental immunology

Article Title: Targeting C3a/C5a receptors inhibits human mesangial cell proliferation and alleviates immunoglobulin A nephropathy in mice.

doi: 10.1111/cei.12961

Figure Lengend Snippet: Fig. 2. Wild-type (WT), C3aR deficient (C3aR–/–) and C5aR–/– mice were immunized with inactivated Sendai virus to induce immunoglobulin (Ig)A nephropathy (IgAN). After 14 weeks of immunization, IgA and C3 deposition in the mesangium and the associated histological changes were analysed. (a,b) Glomerular IgA (upper row) and C3 (low row) deposition was measured by immunofluorescence staining of kidney sections from WT, C3aR–/– and C5aR–/– IgAN mice and negative controls. (c) Mesangial matrix expansion and cell proliferation were demonstrated by periodic acid-Schiff (PAS) staining of renal tissue in the following four groups of mice: negative controls and WT, C3aR–/– and C5aR–/– IgAN mice. Magnification 3400. Scale bars represent 100 lM.

Article Snippet: The membranes were blocked with 5% skimmed milk solution in 0 1% Tris-buffered saline Tween-20 (Dingguo Bioscience) for 1 h. The membranes were incubated subsequently with either mouse antihuman C3aR monoclonal antibodies (1 : 500 dilution; AbD Serotec, Oxford, UK), mouse anti-human C5aR monoclonal antibodies (1 : 500 dilution; AbD Serotec), rabbit anti-human interleukin (IL)-6 monoclonal antibodies (1 : 800 dilution; Abcam, Cambridge, UK), rabbit antihuman MCP-1 polyclonal antibodies (1:1000 dilution, Abcam) or rabbit anti-human GAPDH polyclonal antibodies (1 : 800 dilution; Good Here Bioscience, Hangzhou, China) overnight at 4 C with gentle shaking.

Techniques: Virus, Immunofluorescence, Staining

Journal: Clinical and experimental immunology

Article Title: Targeting C3a/C5a receptors inhibits human mesangial cell proliferation and alleviates immunoglobulin A nephropathy in mice.

doi: 10.1111/cei.12961

Figure Lengend Snippet: Fig. 3. Representative images of immunohistochemical staining for interleukin (IL)-6 and monocyte chemotactic protein 1 (MCP-1) in the kidney sections of wild-type (WT), C3aR deficient (C3aR–/–) and C5aR–/– immunoglobulin (Ig)A nephropathy (IgAN) mice and negative controls. C3aR and C5aR deficiency reduced renal IL-6 and MCP-1 expression compared to WT mice. Scale bars represent 100 lM.

Article Snippet: The membranes were blocked with 5% skimmed milk solution in 0 1% Tris-buffered saline Tween-20 (Dingguo Bioscience) for 1 h. The membranes were incubated subsequently with either mouse antihuman C3aR monoclonal antibodies (1 : 500 dilution; AbD Serotec, Oxford, UK), mouse anti-human C5aR monoclonal antibodies (1 : 500 dilution; AbD Serotec), rabbit anti-human interleukin (IL)-6 monoclonal antibodies (1 : 800 dilution; Abcam, Cambridge, UK), rabbit antihuman MCP-1 polyclonal antibodies (1:1000 dilution, Abcam) or rabbit anti-human GAPDH polyclonal antibodies (1 : 800 dilution; Good Here Bioscience, Hangzhou, China) overnight at 4 C with gentle shaking.

Techniques: Immunohistochemical staining, Staining, Expressing

Journal: Clinical and experimental immunology

Article Title: Targeting C3a/C5a receptors inhibits human mesangial cell proliferation and alleviates immunoglobulin A nephropathy in mice.

doi: 10.1111/cei.12961

Figure Lengend Snippet: Fig. 4. Cytokine and chemokine gene expression in the renal tissues of the following four groups of mice: negative controls and wild-type (WT), C3aR deficient (C3aR–/–) and C5aR–/– immunoglobulin (Ig)A nephropathy (IgAN) mice (n5 7 for each group). Reverse transcription–quantitative polymerase chain reaction (RT–qPCR) was used to quantify the mRNA expression levels of (a) tumour necrosis factor (TNF)-a, (b) transforming growth factor (TGF)-b, (c) interleukin (IL)-1b, (d) interleukin (IL)-6 and (e) monocyte chemotactic protein 1 (MCP-1) relative to glyceraldehyde-3- phosphate dehydrogenase (GAPDH). Data are expressed as the mean 6 standard deviation. *P< 005, **P< 001, ***P< 0001 versus negative control.

Article Snippet: The membranes were blocked with 5% skimmed milk solution in 0 1% Tris-buffered saline Tween-20 (Dingguo Bioscience) for 1 h. The membranes were incubated subsequently with either mouse antihuman C3aR monoclonal antibodies (1 : 500 dilution; AbD Serotec, Oxford, UK), mouse anti-human C5aR monoclonal antibodies (1 : 500 dilution; AbD Serotec), rabbit anti-human interleukin (IL)-6 monoclonal antibodies (1 : 800 dilution; Abcam, Cambridge, UK), rabbit antihuman MCP-1 polyclonal antibodies (1:1000 dilution, Abcam) or rabbit anti-human GAPDH polyclonal antibodies (1 : 800 dilution; Good Here Bioscience, Hangzhou, China) overnight at 4 C with gentle shaking.

Techniques: Gene Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Standard Deviation, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: C5aR1 signaling triggers lung immunopathology in COVID-19 through neutrophil extracellular traps

doi: 10.1172/JCI163105

Figure Lengend Snippet: An ELISA assay was performed to measure the concentrations of ( A ) C5a, ( B ) factor Bb, and ( C ) C3a in the BAL fluid from patients with influenza ( n = 16) and patients with COVID- 19 ( n = 16). ( B ) Paired concentrations of ( D ) C5a and ( E ) factor Bb in the plasma and BAL fluid from patients with COVID-19 were determined by ELISA. ( F ) A different cohort from a previously published data set was reanalyzed and the t-SNE analysis of total cells (65,166) from BAL fluid of patients with non-COVID-19 pneumonia ( n = 13) and COVID-19 ( n = 22) is shown. ( G ) Dot plots display the highlighted distribution of C5AR1 for each indicated cell population. ( H ) Violin plots showing the expression levels of C5aR1 in each type of cell from patients with COVID-19 or with non-COVID-19 pneumonia. ( I ) The dot plot depicts the scaled and centered expression of an average cell in each cluster and therefore contains negative and positive values. The average expression reflects the mean expression of C5AR1 in each cluster compared with all other cells. ( J ) Number of cells per cell population [neutrophils (Neu), monocytes/macrophages (Mo/Mac), and dendritic cells (cDC)] that express C5AR1 in the groups of patients with COVID-19 and non-COVID-19 pneumonia. ( K ) Average expression of C5AR1 per cell for each cell population [neutrophils (Neu), monocytes/macrophages (Mo/Mac), and dendritic cells (cDC)] in the groups of patients with COVID-19 and non-COVID-19 pneumonia. Data are shown as the mean ± SEM. P values were determined by 2-tailed unpaired ( A – D , J , and K ) or paired ( D and E ) Student’s t tests followed by Wilcoxon matched-pairs signed rank tests.

Article Snippet: C5a, factor Bb, and C3a ELISA assays were performed using, respectively, the human complement component C5a duoset ELISA kit from R&D Systems (DY2037), the MicroVue Bb plus fragment EIA kit from Quidel (A027), and the Complement C3a human ELISA kit from Thermo Fisher Scientific (BMS2089).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Expressing

Journal: The Journal of Clinical Investigation

Article Title: C5aR1 signaling triggers lung immunopathology in COVID-19 through neutrophil extracellular traps

doi: 10.1172/JCI163105

Figure Lengend Snippet: ( A ) Tg mice were infected with SARS-CoV-2 (2 × 104 PFU, intranasally). ELISA assay to measure levels of ( B ) C5a in the lung homogenate of infected animals ( n = 14) or mock control ( n = 11). ( C ) factor Bb and ( D ) C3a levels in the lung homogenate of infected animals ( n = 8) or mock control ( n = 5). ( E ) Representative confocal images of the presence of C5aR1 expression in the lung tissue of Tg fl/fl mice ( C5ar1 -eGFP mice) infected with SARS-CoV-2 (5 dpi). Tissue slices were costained for nuclei (DAPI, blue), Iba-1 (macrophages, red) and NE (neutrophils, red) markers. Scale bar: 50 μm. ( F ) Percentage of cells expressing C5aR1 in the lung tissue of Tg fl/fl mice infected with SARS-CoV-2 ( n = 4 mice/4 randomized field). ( G ) Representative H&E staining from the lung of SARS-CoV-2-infected Tg fl/fl ( n = 6) or Tg cKO mice ( n = 6). A mock-infected group was used as control ( n = 6). Scale bars: 200 μm (4 ×), 100 μm(10 ×). ( H ) TUNEL staining (red) for detection of apoptotic cells in situ from lung tissue of SARS-CoV-2–infected Tg fl/fl ( n = 5) or Tg cKO mice ( n = 6). Mock-infected Tg mice were used as a control ( n = 5/group). ( I ) Quantification of the lung septal area fraction. ( J ) Percentage of TUNEL positive cells in lung tissue. Scale bar: 50 μm. ( K ) ELISA assays were performed to detect CCL2, CCL3, CCL4, CXCL1, and IL-6 levels in the lung tissue of Tg fl/f ( n = 8) or Infected Tg cKO mice ( n = 7). Mock-infected Tg mice were used as a control ( n = 5). Data are shown as the mean ± SEM. P values were determined by ( B – D ) Student’s 2-tailed t test and ( F , I , J , and K ) 1-way ANOVA followed by Bonferroni’s posthoc test.

Article Snippet: C5a, factor Bb, and C3a ELISA assays were performed using, respectively, the human complement component C5a duoset ELISA kit from R&D Systems (DY2037), the MicroVue Bb plus fragment EIA kit from Quidel (A027), and the Complement C3a human ELISA kit from Thermo Fisher Scientific (BMS2089).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Control, Expressing, Staining, TUNEL Assay, In Situ

Journal: The Journal of Clinical Investigation

Article Title: C5aR1 signaling triggers lung immunopathology in COVID-19 through neutrophil extracellular traps

doi: 10.1172/JCI163105

Figure Lengend Snippet: ( A ) Tg mice were infected with SARS-CoV-2 (2 x 104 PFU, intranasally) and treated with DF2593A (3 mg/kg, p.o) 1 hour before SARS-CoV-2 infection and once a day up to the day of sample collection (5 dpi). ( B ) Body weight, clinical score, and oxygen saturation were measured daily after infection ( n = 11/group, pooled from 2 independent experiments). ( C ) Representative H&E staining from the harvested lung of the COVID-19 mouse model treated ( n = 4) or not ( n = 6) with DF2593A. A mock-infected group was used as control ( n = 5). Scale bars: 200 μm (4 ×); 100 μm (10 ×). ( D ) TUNEL staining (green) for detection of apoptotic cells in situ from lung tissue of mice ( n = 5/group). ( E ) Quantification of the lung septal area fraction. ( F ) Percentage of TUNEL-positive cells in lung tissue. Scale bar: 50 μm. ( G ) ELISA assays were performed to detect CCL2, CCL3, CCL4, CXCL1, and IL-6 levels in lung homogenate ( n = 6/group). A mock-infected group was used as the control group. Data are shown as the mean ± SEM. P values were determined by 1-way ANOVA followed by Bonferroni’s posthoc test ( E – G ).

Article Snippet: C5a, factor Bb, and C3a ELISA assays were performed using, respectively, the human complement component C5a duoset ELISA kit from R&D Systems (DY2037), the MicroVue Bb plus fragment EIA kit from Quidel (A027), and the Complement C3a human ELISA kit from Thermo Fisher Scientific (BMS2089).

Techniques: Infection, Staining, Control, TUNEL Assay, In Situ, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: C5aR1 signaling triggers lung immunopathology in COVID-19 through neutrophil extracellular traps

doi: 10.1172/JCI163105

Figure Lengend Snippet: ( A ) C57/BL6 mice were treated twice with vehicle, DNAse (Pulmozyme, 10 mg/kg, s.c.), or C5aR1 antagonist (DF2593A, 3 mg/kg, orally), 24 hours and 1 hour before the intratracheal instillation of rmC5a (400 ng). ( B ) Lung slices from the control group or mice challenged with rmC5a and treated with vehicle, DNAse, or C5aR1 antagonist (DF2593A) were stained with H&E for evaluation of histological changes. ( C ) Quantification of the lung septal area fraction ( n = 5/group). ( D ) Lung slices from the control group or from mice challenged with rmC5a and treated with vehicle, DNAse, or C5aR1 antagonist (DF2593A) were costained for nuclei (DAPI, blue), H3Cit (green), and MPO (red) markers. ( E ) NET quantification by the MPO-DNA PicoGreen assay in the supernatant of the lung homogenate ( n = 5–6/group). ( F ) ELISA assays were performed to detect CCL2, CCL3, CCL4, CXCL1, and IL-6 levels in lung homogenate ( n = 5–6/group). Data are shown as the mean ± SEM. P values were determined by 1-way ANOVA followed by Bonferroni’s posthoc test ( C , E , and F ).

Article Snippet: C5a, factor Bb, and C3a ELISA assays were performed using, respectively, the human complement component C5a duoset ELISA kit from R&D Systems (DY2037), the MicroVue Bb plus fragment EIA kit from Quidel (A027), and the Complement C3a human ELISA kit from Thermo Fisher Scientific (BMS2089).

Techniques: Control, Staining, Picogreen Assay, Enzyme-linked Immunosorbent Assay

Journal: BMC Genomics

Article Title: Transcriptional profiling reveals developmental relationship and distinct biological functions of CD16+ and CD16- monocyte subsets

doi: 10.1186/1471-2164-10-403

Figure Lengend Snippet: Real-time RT-PCR validation of microarray results . The expression of CD16, C3AR1, C1QR1, ICAM-2, CSFR1, CSF3R, CDKN1C, TNFRSF8, and LTB mRNA was quantified by SYBR Green real time RT-PCR in CD16 + and CD16 - Mo. The concentration of each gene was normalized to the 28S rRNA internal control and expressed as fgs RNA of a target gene per 1 ng rRNA28S. Depicted are results (mean ± SD of triplicate wells; *, p < 0.05, unpaired t-test, CD16 + versus CD16 - Mo) obtained with matched cells from 2 different healthy donors.

Article Snippet: Fluorochrome-conjugated Abs used for FACS analysis were CD14, CD16, CD19, CD16b, CD66b, CD56, and CD3 (Beckman Coulter); M-DC8 and CD1c (Miltenyi), HLA-DR, CD114, C3aR, CD1d and CD43 (BD Pharmingen), CD115 (R&D Systems), CD93/C1qR1 (Chemicon International), and CXCL16 (R&D Systems).

Techniques: Quantitative RT-PCR, Biomarker Discovery, Microarray, Expressing, SYBR Green Assay, Concentration Assay, Control

Journal: BMC Genomics

Article Title: Transcriptional profiling reveals developmental relationship and distinct biological functions of CD16+ and CD16- monocyte subsets

doi: 10.1186/1471-2164-10-403

Figure Lengend Snippet: Differential expression of CD114/CSF3R, CD115/CSF1R, CD93/C1qR1 and C3aR1 on CD16 + and CD16 - monocytes . Freshly isolated PBMC were stained with FITC CD14, PE-Cy5 CD16, and PE CD114, PE CD115, and PE CD93 Abs. The expression of CD3aR1 was detected after staining with unconjugated mouse C3AR1 Ab and PE rat anti-mouse Ab (RAM). CD14 high CD16 neg (R2) and CD14 low CD16 + (R3) Mo (A) were analyzed for expression of CD114, CD115, CD93 and C3aR1 (B) . Shown is an overlay histogram from one representative donor of 4 donors examined (B, left panels) and graphs showing mean ± SEM for % or MFI of CD114, CD115, CD93 and C3aR1 expression on each Mo subset (B, right panels) . (*, Paired t-test p-value < 0.05, CD16 + versus CD16 - Mo; n = 4).

Article Snippet: Fluorochrome-conjugated Abs used for FACS analysis were CD14, CD16, CD19, CD16b, CD66b, CD56, and CD3 (Beckman Coulter); M-DC8 and CD1c (Miltenyi), HLA-DR, CD114, C3aR, CD1d and CD43 (BD Pharmingen), CD115 (R&D Systems), CD93/C1qR1 (Chemicon International), and CXCL16 (R&D Systems).

Techniques: Quantitative Proteomics, Isolation, Staining, Expressing

Journal: Frontiers in Immunology

Article Title: Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices

doi: 10.3389/fimmu.2021.777932

Figure Lengend Snippet: Microfluidic devices for measuring neutrophil chemotaxis, phagocytosis, and swarming behaviors. (A) Microscopic image of a segment of the chemotaxis microfluidic device (x10 Brightfield, Nikon TiE) showing neutrophil chemotaxis towards C5a and C3a during increasing mechanical restriction. Each chemoattractant chamber is connected to the cell loading channel through 3 tapered channels. (B) Microscopic image of a segment of the phagocytosis microfluidic device (x10 Brightfield/Fluorescent, Nikon TiE) showing neutrophils (blue nuclei) from the outer chamber migrating towards the central reservoir through a migration channel to phagocytose S. aureus particles (green) in plasma at T 20 minutes. (C) Microscopic images obtained at three different time points (T 0 , T 10 , and T 30 minutes) showing the formation of a neutrophil swarm (blue nuclei) over a cluster of Texas red-labeled zymosan A S. cerevisiae bioparticles (Red).

Article Snippet: The chemoattractant chambers were filled either with fMLP (N-formyl-methionyl-leucyl-phenylalanine, 100 nM, Millipore Sigma), recombinant human C5a protein (ab61918, 0.1 - 10 μM, Abcam), or C3a (3677-C3-025, 0.1 - 10 μM, R&D Systems) diluted in HBSS with 0.5% BSA during the initial priming step.

Techniques: Chemotaxis Assay, Migration, Clinical Proteomics, Labeling

Journal: Frontiers in Immunology

Article Title: Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices

doi: 10.3389/fimmu.2021.777932

Figure Lengend Snippet: Chemotaxis of isolated neutrophils towards C5a and C3a and the effect of AVA on chemotaxis. (A) The percentage of neutrophils migrating directionally towards increasing concentrations of C5a and C3a compared to media alone (HBSS with 0.5%BSA, N=3, ****p < 0.0001; One-way ANOVA with Dunnett’s test) (B) C3a inhibits neutrophil chemotaxis towards C5a, C5a effect is dependent on concentration, and there is no interaction effect between C3 and C5a. (N=3 donors, p < 0.005, Repeated measures, two way ANOVA, with Sidak’s correction for multiple comparisons). (C) Percentage of neutrophils migrating directionally towards C5a (100 nM) after treatment of neutrophils with C5aR1 antagonist AVA (N=3, 4 or 5, each dot on the plot represents one experiment, ****p < 0.01 for comparisons to C5a control; ns represents not statistical significant. One way ANOVA with Dunnett’s multiple comparison test with single pooled variance).

Article Snippet: The chemoattractant chambers were filled either with fMLP (N-formyl-methionyl-leucyl-phenylalanine, 100 nM, Millipore Sigma), recombinant human C5a protein (ab61918, 0.1 - 10 μM, Abcam), or C3a (3677-C3-025, 0.1 - 10 μM, R&D Systems) diluted in HBSS with 0.5% BSA during the initial priming step.

Techniques: Chemotaxis Assay, Isolation, Concentration Assay, Control, Comparison

Journal: Frontiers in Immunology

Article Title: Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices

doi: 10.3389/fimmu.2021.777932

Figure Lengend Snippet: Atypical membrane elasticity of neutrophils and migration patterns when exposed to high C5a dose. (A) Percentage of neutrophil migration patterns during chemotaxis towards C5a, C3a, and fMLP. (N=3). (B) Microscopic images of elongated neutrophils when exposed to C5a 1µM inside the end chambers. Scale = 100 µm. (C) Percentages of unique reversed migration phenotype of neutrophils observed when exposed to high C5a concentration. (N=3, *p < 0.05; ns represents not statistical significant. One-way ANOVA with Dunnett’s test).

Article Snippet: The chemoattractant chambers were filled either with fMLP (N-formyl-methionyl-leucyl-phenylalanine, 100 nM, Millipore Sigma), recombinant human C5a protein (ab61918, 0.1 - 10 μM, Abcam), or C3a (3677-C3-025, 0.1 - 10 μM, R&D Systems) diluted in HBSS with 0.5% BSA during the initial priming step.

Techniques: Membrane, Migration, Chemotaxis Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices

doi: 10.3389/fimmu.2021.777932

Figure Lengend Snippet: Effect of C5 complement inhibitors on neutrophil chemotaxis towards endogenous complement activation with CVF. (A) Effect of AVA (250 nM) on endogenous complement activation by increasing CVF doses (N=3 donors, *p < 0.05, **p < 0.01; paired two-tailed t -test). (B) Effect of C5 inhibition with ECU (3 µM) and RA101295 (3 µM) on endogenous complement activation by CVF (N=3, **p < 0.01; ns represents not statistical significant. One-way ANOVA with Dunnett’s test).

Article Snippet: The chemoattractant chambers were filled either with fMLP (N-formyl-methionyl-leucyl-phenylalanine, 100 nM, Millipore Sigma), recombinant human C5a protein (ab61918, 0.1 - 10 μM, Abcam), or C3a (3677-C3-025, 0.1 - 10 μM, R&D Systems) diluted in HBSS with 0.5% BSA during the initial priming step.

Techniques: Chemotaxis Assay, Activation Assay, Two Tailed Test, Inhibition

Journal: Frontiers in Immunology

Article Title: Human Neutrophils Respond to Complement Activation and Inhibition in Microfluidic Devices

doi: 10.3389/fimmu.2021.777932

Figure Lengend Snippet: Schematic representation of complement activation pathways, terminal inhibition strategies, and their effects on neutrophil functional behaviors. The classical, lectin, and alternative pathways converge on the cleavage of the central components C3 into C3a and C3b peptides. C3a acts as an anti-inflammatory mediator towards neutrophils, while C3b enhances phagocytosis through opsonization and forms C5 convertases, which cleaves C5 into C5a and C5b. C5a is a potent pro-inflammatory mediator and chemoattractant, while C5b forms the membrane attack complex (MAC), leading to cell and bacteria lysis. ECU and RA101295 are C5-inhibitors, which block the production of C5a and C5b peptides, while AVA blocks C5a mediated chemotaxis and pro-inflammatory effects.

Article Snippet: The chemoattractant chambers were filled either with fMLP (N-formyl-methionyl-leucyl-phenylalanine, 100 nM, Millipore Sigma), recombinant human C5a protein (ab61918, 0.1 - 10 μM, Abcam), or C3a (3677-C3-025, 0.1 - 10 μM, R&D Systems) diluted in HBSS with 0.5% BSA during the initial priming step.

Techniques: Activation Assay, Inhibition, Functional Assay, Membrane, Bacteria, Lysis, Blocking Assay, Chemotaxis Assay