Journal: Biochemistry and biophysics reports

Article Title: Complement dependent TNFα production in neutrophil-like HL60 cells.

doi: 10.1016/j.bbrep.2023.101465

Figure Lengend Snippet: Fig. 2. Complement-mediated TNFα production in neutrophil-like HL60 cells. (A) Zymosan particles were incubated in human serum that was either non-treated (SOZ) or heat- inactivated (HI-SOZ) or pre-treated with compstatin (Comp) or control peptide (CTRL). Non-treatment (NT) indicates zymosan particles that were not treated with human serum and any reagent. Binding of C3bi to zymosan was analyzed by flow cytometry with anti-C3bi antibody. Representative histograms (left) and bar graph of Median ratio = Median fluo rescence intensity for each sample/Median fluores cence intensity for Without 1st Ab (right) at the indicated conditions are presented. Data show the means ± SD derived from 4 independent experiments (n = 4). p values were calculated using a two-tailed unpaired Student’s t-test. ** <0.01. (B) Day 5 ATRA-treated HL60 cells were incubated with 10% human serum pre-treated under the conditions indi cated for 4 h, and the whole cell lysates (Cell) and culture supernatants (Sup.) were then collected. Non- treatment (NT) indicates cells that were not treated with human serum and any reagent. TNFα and β-actin in samples were detected by immunoblot analysis, as shown in Fig. 1. RPMI, Human serum, and ZHS in diagonal letters indicate samples of RPMI-1640 me dium, RPMI-1640 containing ZHS, or HS with 1 × SDS-PAGE sample loading buffer, respectively. The blot is a representative of 3 independent experiments.

Article Snippet: Compstatin (#2585) and compstatin control peptide (#3796) were purchased from Tocris Bioscience (Abingdon, UK).

Techniques: Incubation, Control, Binding Assay, Flow Cytometry, Derivative Assay, Two Tailed Test, Western Blot, SDS Page

Journal: Frontiers in immunology

Article Title: Complement C3b contributes to Escherichia coli -induced platelet aggregation in human whole blood.

doi: 10.3389/fimmu.2022.1020712

Figure Lengend Snippet: FIGURE 1 Escherichia coli (E. coli, 5.3 x 109/mL)-induced platelet aggregation measured by Multiplate® impedance aggregometry. To the samples were added phosphate-buffered saline (PBS) control, eculizumab (Eculiz, 100 µg/mL), compstatin (Cp40, 20 µM), anti-CD14 antibody (aCD14, 15 µg/mL), immunoglobulin G 2/4 control antibody (NHDL, 15 µg/mL) and control peptide (ctrl.peptide, 20 µM) as inhibitors, and PBS or E. coli as activators. Results are given as the area under the curve (AUC), using mean ± standard deviation (n = 6). #; p < 0.05 analyzed using a paired Student’s t-test between the samples with and without E. coli, *; p < 0.05 analyzed using one-way ANOVA repeated measurements, Dunnett’s multiple comparisons test, comparing E. coli-activated samples with and without inhibitors.

Article Snippet: The compstatin control peptide (R&D Systems), a LEAF TM Purified mouse IgG1k (Biolegend), and polyclonal sheep IgG (R&D Systems) were used as controls.

Techniques: Saline, Control, Standard Deviation

Journal: Frontiers in immunology

Article Title: Complement C3b contributes to Escherichia coli -induced platelet aggregation in human whole blood.

doi: 10.3389/fimmu.2022.1020712

Figure Lengend Snippet: FIGURE 2 The effect of C3a and C3b (C3a, C3a receptor antagonist (RA), anti-human C3a antibody and blocking anti-human C3b antibody) on Escherichia coli (E. coli 5.3 x 109/mL)-induced platelet aggregation measured by Multiplate® impedance aggregometry. To the samples were added phosphate-buffered saline (PBS) control or purified C3a in increasing concentrations (200 nM and 400 nM) (n = 5) (A). To the samples were added PBS control or C3aRA in increasing concentrations (10 nM, 100 nM and 1000 nM) (n = 4) (B). To the samples were added PBS control, C3a antibody (aC3a, 0.2 g/mL), compstatin (Cp40, 20 µM) or control antibody (ctrl.Ab, 0.2 mg/mL) (n = 5) (C). To the samples were added PBS control, C3b antibody (aC3b 0.2 mg/mL), compstatin (Cp40, 20 µM) or control antibody (ctrl.Ab 0.2 mg/mL) (n = 3) (D). To all samples, PBS or E. coli were added as activators. Results are given as the area under the curve (AUC), using mean ± standard deviation. #; p < 0.05 analyzed using a paired Student’s t-test between the samples with and without E. coli, *; p < 0.05 analyzed using one-way ANOVA repeated measurements, and Dunnett’s multiple comparisons test, comparing E. coli-activated samples with and without inhibitors or control.

Article Snippet: The compstatin control peptide (R&D Systems), a LEAF TM Purified mouse IgG1k (Biolegend), and polyclonal sheep IgG (R&D Systems) were used as controls.

Techniques: Blocking Assay, Saline, Control, Standard Deviation

Journal: Frontiers in immunology

Article Title: Complement C3b contributes to Escherichia coli -induced platelet aggregation in human whole blood.

doi: 10.3389/fimmu.2022.1020712

Figure Lengend Snippet: FIGURE 3 The C3b opsonization of platelets and Escherichia coli (E. coli 1 x 109/mL) were measured by flow cytometry after performing the human whole blood model. E. coli was labeled with Alexa Fluor 633, platelets were detected by BV605-labeled anti-CD61-antibody, leukocytes by V450- labeled anti-CD45-antibody, and C3b opsonization was measured using a FITC-labeled anti-C3c-antibody. The blood was incubated at 37 °C for 10 minutes after adding phosphate-buffered saline (PBS) control, compstatin (Cp40, 20 µM) or control peptide (20 µM), and PBS or E. coli as activators. The population of all platelets, both platelets alone and in conjugation with E. coli, was gated in an aCD45-aCD61 plot (A). The Alexa 633-aCD61 plot was used to separate the platelets alone (red) and the platelets in conjugation with E. coli (blue) (B). Histogram showing the C3b opsonization of platelet-E. coli conjugates; the sample without inhibition in blue, the sample with compstatin in gray (C). Histogram showing the C3b opsonization of the platelets alone; the sample without inhibition in red and the sample added compstatin in gray (D). The plots (A-D) were from one of the six donors and were representative of the other donors which showed the same pattern. E. coli-induced C3b opsonization is shown in a dot plot diagram (E), with data for platelets alone (closed circles) and data for platelets together with E. coli (open circles) (n = 6). Results are given in mean fluorescence (MFI) for C3c, the mean is reported as lines in the figure. *; p < 0.05 analyzed using one- way ANOVA repeated measurements, and Dunnett’s multiple comparisons test, comparing E. coli-activated samples with and without inhibitors or control. A confocal super-resolution image of platelets interacting with C3b opsonized E. coli in whole blood (F). Platelets were detected with a BV605-labeled anti-CD61 antibody (red), and C3b was detected with a FITC-labeled anti-C3c antibody (green).

Article Snippet: The compstatin control peptide (R&D Systems), a LEAF TM Purified mouse IgG1k (Biolegend), and polyclonal sheep IgG (R&D Systems) were used as controls.

Techniques: Cytometry, Labeling, Incubation, Saline, Control, Conjugation Assay, Inhibition

Journal: Frontiers in immunology

Article Title: Complement C3b contributes to Escherichia coli -induced platelet aggregation in human whole blood.

doi: 10.3389/fimmu.2022.1020712

Figure Lengend Snippet: FIGURE 5 Platelet aggregation with isolated platelets in Tyrode’s buffer was measured by Multiplate® impedance aggregometry. The platelets were isolated by centrifugation and washed using Tyrode’s buffer. The samples were added phosphate-buffered saline (PBS) or Escherichia coli (E. coli) in PBS in increasing concentrations as indicated; 1 x 107/mL, 1 x 108/mL, 1 x 109/mL, and 1 x 1010/mL, n = 3 (A). *; p < 0.05 analyzed using one-way repeated measurements ANOVA, and Dunnett’s multiple comparisons test, comparing samples with increasing E. coli concentrations to the lowest E. coli concentration (1 x 107/mL). The effect of C3b on aggregation of isolated platelets were analyzed (B). To the samples were either added PBS, C3b (20 nM), compstatin (Cp40, 20 µM) or a combination of them. Adenosine diphosphate (ADP) was added as a positive control. The results are given as the area under the curve (AUC), using mean ± standard deviation (n = 5). *; p < 0.05 analyzed using one-way ANOVA repeated measurements, and Dunnett’s multiple comparisons test, comparing the PBS control to the other samples.

Article Snippet: The compstatin control peptide (R&D Systems), a LEAF TM Purified mouse IgG1k (Biolegend), and polyclonal sheep IgG (R&D Systems) were used as controls.

Techniques: Isolation, Centrifugation, Saline, Concentration Assay, Positive Control, Standard Deviation, Control

Journal: Frontiers in immunology

Article Title: Complement C3b contributes to Escherichia coli -induced platelet aggregation in human whole blood.

doi: 10.3389/fimmu.2022.1020712

Figure Lengend Snippet: FIGURE 6 The effect of a blocking complement receptor (CR)1 antibody on Escherichia coli (E. coli 5.3 x 109/mL)-induced platelet aggregation measured on Multiplate® impedance aggregometry. To the samples were added phosphate-buffered saline (PBS) control, compstatin (Cp40, 20 µM), blocking CR1 antibody (4 ng/ mL) or a combination of both, and PBS or E. coli as activators. Results are given as the area under the curve (AUC), using mean ± standard deviation (n = 7). #; p < 0.05 analyzed using a paired Student’s t-test between the samples with and without E. coli, *; p < 0.05 analyzed using one-way ANOVA repeated measurements, and Dunnett’s multiple comparisons test, comparing E. coli-activated samples with and without inhibitors.

Article Snippet: The compstatin control peptide (R&D Systems), a LEAF TM Purified mouse IgG1k (Biolegend), and polyclonal sheep IgG (R&D Systems) were used as controls.

Techniques: Blocking Assay, Saline, Control, Standard Deviation

Journal: Frontiers in immunology

Article Title: Complement C3b contributes to Escherichia coli -induced platelet aggregation in human whole blood.

doi: 10.3389/fimmu.2022.1020712

Figure Lengend Snippet: FIGURE 7 The role of glycoprotein (GP) IIb/IIIa on Escherichia coli (E. coli 5.3 x 109/mL)-induced platelet aggregation measured by Multiplate® impedance aggregometry (A). To the samples were added phosphate-buffered saline (PBS) control, tirofiban (5 µg/mL), compstatin (Cp40, 20 µM) or the combination of tirofiban and compstatin, and PBS or E. coli as activators. The results are given as the area under the curve (AUC), using median ± interquartile range (n = 6). The effect of C3-inhibition on the E. coli-induced (1 x 109/mL) activated GPIIb/IIIa on platelets measured by flow cytometry (B). To the samples were added PBS control, tirofiban (5 µg/mL), compstatin (Cp40, 20 µM), or control peptide (ctrl.peptide, 20 µM), and PBS or E. coli as activators. A BV605-labeled anti-human CD61 antibody was used to gate the platelets and a FITC-labeled anti-human PAC-1 antibody was used to detect the activated GPIIb/IIIa receptors. Results are expressed as percent of the uninhibited sample (E. coli without inhibition set to 100%) and are given as median ± interquartile range (n = 16). #; p < 0.05 analyzed using a Wilcoxon test between the samples with and without E. coli, *; p < 0.05 analyzed using a Friedman test and Dunn’s multiple comparisons test, comparing E. coli-activated samples without and with inhibition or control. §; p < 0.05 analyzed using a Wilcoxon test between the E. coli-activated samples with compstatin (Cp40) and with the combination of compstatin and tirofiban.

Article Snippet: The compstatin control peptide (R&D Systems), a LEAF TM Purified mouse IgG1k (Biolegend), and polyclonal sheep IgG (R&D Systems) were used as controls.

Techniques: Saline, Control, Inhibition, Cytometry, Labeling