Journal: Scientific reports

Article Title: Complement classical and alternative pathway activation contributes to diabetic kidney disease progression: a glomerular proteomics on kidney biopsies.

doi: 10.1038/s41598-024-84900-4

Figure Lengend Snippet: Fig. 3. Immunohistochemistry (IHC) staining for C1q, C3, C4, C5b-9, CFB, CFH, MBL, and MASP. (a–e) Representative images of C1q, C3, C4, C5b-9, and CFB staining showing a GBM pattern of immunostaining in kidney tissues from patients with DKD. (f) Immunohistochemical diagram of CFH in kidneys with DKD depicting mesangial deposition. (g, h) Representative images of MBL and MASP staining showing no deposition was found in the glomeruli of kidneys with DKD. Scale bars represent 50 μm. CFB, complement factor B; CFH, complement factor H; MBL, mannose-binding lectin; MASP, mannose-binding lectin- associated serine protease; GBM, glomerular basement membrane; DKD, diabetic kidney disease.

Article Snippet: After washing, sections were incubated with either anti-C1q antibodies (1:200 dilution, 16889-1-AP; Proteintech), anti-C3 antibodies (1:200 dilution, ab200999; Abcam), anti-C4 antibodies (1:300 dilution, 22233-1-AP; Proteintech), anti-C5b-9 antibodies (1:400 dilution, ab55811; Abcam), anti-MBL antibodies (1:200 dilution, ab23457; Abcam), anti-MASP1 antibodies (1:400 dilution, 21837-1-AP; Proteintech), anti-CFB antibodies (1:50 dilution, ab192577; Abcam), or anti-CFH antibodies (1:50 dilution, ab170036; Abcam) overnight at 4 °C in a moist chamber.

Techniques: Immunohistochemistry, Staining, Immunostaining, Immunohistochemical staining, Binding Assay, Membrane

Journal: Bioactive Materials

Article Title: Engineered neutrophil apoptotic bodies ameliorate myocardial infarction by promoting macrophage efferocytosis and inflammation resolution

doi: 10.1016/j.bioactmat.2021.08.008

Figure Lengend Snippet: Preparation and characterization of Neu-ABs and eNABs. ( A ) Schematic shows the purification of Neu-ABs through apoptosis induction and serial centrifugations, as well as the generation of eNABs. The latter process was conducted using a liposome extruder and followed two steps: (i) NABM was generated by repeated extrusion through the first filter (pore diameter: 400 nm); (ii) eNABs were generated through repeated extrusion of the mixed suspension (NABM and MSNs) through a smaller filter (pore diameter: 200 nm). ( B ) Representative SEM image of ABs derived from neutrophils. Scale bars, 1 μm. ( C ) The Annexin V and C1q staining of Neu-ABs. Scale bars, 5 μm. ( D ) Western blotting analysis of neutrophil and Neu-ABs. ( E ) Representative TEM images of MSNs and eNABs. Scale bars, 50 nm. ( F ) Size distribution of NABM, MSNs, and eNABs measured by DLS. Average diameter ( G ) and surface zeta potential ( H ) of NABM, MSNs, and eNABs (n = 3). ( I ) Representative images of the fluorescence colocalization of PKH67-labeled NABM and rhodamine-labeled MSNs in eNABs. The pure rhodamine-labeled MSNs were used as a control. Scale bars, 10 μm. ( J ) SDS-PAGE protein analysis of NABM, MSNs, and eNABs with Coomassie Blue staining. ( K ) Western blot analysis for membrane-specific protein markers. The storage stability ( L ) and the serum stability ( M ) of eNABs (n = 3). Data are presented as means ± SD. Statistical analysis between two groups was performed using unpaired two-tailed t-tests for M (** P < 0.01).

Article Snippet: Fluorescence staining and flow cytometry analysis of ABs was conducted using an Annexin V-FITC apoptosis assay kit (A005-2, 7 Sea Biotech, China) and anti -C1q antibody (CL7501F, CEDARLANE).

Techniques: Purification, Generated, Suspension, Derivative Assay, Staining, Western Blot, Zeta Potential Analyzer, Fluorescence, Labeling, Control, SDS Page, Membrane, Two Tailed Test

Journal: PLoS ONE

Article Title: Alternative Complement Pathway Deregulation Is Correlated with Dengue Severity

doi: 10.1371/journal.pone.0006782

Figure Lengend Snippet: Levels of different components of the complement system in plasma samples collected from dengue-infected patients with different clinical manifestations and at different stages of infection.

Article Snippet: Plasma levels of complement factor D, anaphylotoxins, MBL oligomers, and total IgG immunocomplexes (CIC-C1q) were measured using the Duo Set ELISA development kit for human factor D (R&D systems), the Cytometric Bead Array kit for human anaphylotoxin (BD Biosciences), the MBL oligomer ELISA kit (Innate Immunity Diagnostics), and the CIC-C1q ELISA kit (Quidel), respectively, according to manufacturer directions.

Techniques: Clinical Proteomics, Infection

Journal: PLoS ONE

Article Title: Alternative Complement Pathway Deregulation Is Correlated with Dengue Severity

doi: 10.1371/journal.pone.0006782

Figure Lengend Snippet: Correlations between individual components of the complement system and platelet count in dengue-infected patients during the acute phase.

Article Snippet: Plasma levels of complement factor D, anaphylotoxins, MBL oligomers, and total IgG immunocomplexes (CIC-C1q) were measured using the Duo Set ELISA development kit for human factor D (R&D systems), the Cytometric Bead Array kit for human anaphylotoxin (BD Biosciences), the MBL oligomer ELISA kit (Innate Immunity Diagnostics), and the CIC-C1q ELISA kit (Quidel), respectively, according to manufacturer directions.

Techniques:

Journal: Pathogens

Article Title: Echinococcus multilocularis Calreticulin Inhibits Lectin Pathway of Complement Activation by Directly Binding to Mannose-Binding Lectin

doi: 10.3390/pathogens14040354

Figure Lengend Snippet: Figure 5. rEmCRT and its MBL binding regions inhibited binding of MBL to mannan. (A) Pre- incubation of different amounts of rEmCRT, rEmCRT-S, and rEmCRT-NP (0, 0.5, 1.0 µM) with MBL (10 µg/mL) before adding to plates coated with mannan (50 µg/mL), detected with anti-MBL monoclonal antibody. (B) Under the same ELISA condition, different amounts of rEmCRT, rEmCRT-S, or rEmCRT-NP (0, 1, 2 µM) were pre-incubated with C1qD (1:50) before adding into plates coated with mannan. Data are expressed as mean of OD450 ± SDs of three independent experiments (* p < 0.005; ** p < 0.001, *** p < 0.0005, ns, not significantly different compared to the reading from MBL without incubation with recombinant EmCRT).

Article Snippet: C1q-depleted sera (C1qD) were purchased from Quidel (San Diego, CA, USA).

Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Pathogens

Article Title: Echinococcus multilocularis Calreticulin Inhibits Lectin Pathway of Complement Activation by Directly Binding to Mannose-Binding Lectin

doi: 10.3390/pathogens14040354

Figure Lengend Snippet: Figure 6. Inhibition of C3b and C4b deposition by rEmCRT and its functional binding regions measured by ELISA. (A) Pre-incubation of 100 µL C1qD (1:50) as source of natural MBL with different amounts of rEmCRT, rEmCRT-S, or rEmCRT-NP (0, 2, 4 µM) before adding into mannan-coated plates (50 µg/mL). After being washed, C1qD (1:150) was added as source of complement components to initiate lectin pathway of complement activation. Deposition of C3b (a) and C4b (b) was detected with anti-C3 or C4 monoclonal antibodies. (B) The similar ELISA procedure was performed using NHS as source of physiological MBL instead of C1qD serum to detect the generation of C3b (a) and C4b (b). Data are expressed as mean of OD450 ± SDs of three independent experiments (* p < 0.005, ** p < 0.001, *** p < 0.0005, ns, no significant difference compared to plates without added rEmCRT).

Article Snippet: C1q-depleted sera (C1qD) were purchased from Quidel (San Diego, CA, USA).

Techniques: Inhibition, Functional Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Incubation, Activation Assay, Bioprocessing

Journal: Cell Cycle

Article Title: Blocking Notch signal pathway suppresses the activation of neurotoxic A1 astrocytes after spinal cord injury

doi: 10.1080/15384101.2019.1667189

Figure Lengend Snippet: Real-time reverse transcription polymerase chain reaction (RT-qPCR)

Article Snippet: ELISA kits for TNF-α and IL-1α were purchased from Abcam (San Francisco, CA, USA) and a C1q ELISA kit from Hycult Biotech (Plymouth Meeting, PA, USA).

Techniques: Reverse Transcription, Polymerase Chain Reaction