anti human cd59  (Bio-Rad)

Journal: Frontiers in Medicine

Article Title: Identification of CD320, SLC44A1 and TNFRSF13B as potential novel therapeutic targets for CAR T-cell therapy in multiple myeloma

doi: 10.3389/fmed.2025.1737919

Figure Lengend Snippet: Gene expression and cytogenetic clusters in MM. (A) Violin plots showing gene expression levels for TNFRSF13B , CD59 , FCGR2B , SLC44A1 , and CD320 in HD, MGUS, SMM, MM, RRMM. Statistical comparisons were conducted using the Kruskal–Wallis test for all pairwise contrasts. (B) Heatmap of the pseudobulk expression profiles showing normalized expression levels for eight putative proxy markers of cytogenetic aberrations across 54 MM patients. (C) Box plots illustrating the differential expression of TNFRSF13B , CD59 , FCGR2B , SLC44A1 , and CD320 across the four cytogenetic patient clusters. Statistical significance was evaluated using two-sided t -tests for all pairwise comparisons p < 0.05 (*), p < 0.01 (**), p < 0.001 (***), p < 0.0001 (****).

Article Snippet: Antibodies included: anti-human CD45 V500 (HI30), anti-human CD19 APC-H7 (HIB19), anti-human CD59 FITC (H19), anti-human CD92 BV605 (VIM15), anti-human CD267 BV421 (1A1-K21-M22) (all from BD Biosciences, USA), anti-human CD38 PE (REA572, Miltenyi Biotec, Germany), and anti-human CD320 AlexaFluor 647 (Antibodies Online, Germany).

Techniques: Gene Expression, Expressing, Quantitative Proteomics

Journal: Frontiers in Medicine

Article Title: Identification of CD320, SLC44A1 and TNFRSF13B as potential novel therapeutic targets for CAR T-cell therapy in multiple myeloma

doi: 10.3389/fmed.2025.1737919

Figure Lengend Snippet: Kaplan–Meier survival curves showing the prognostic relevance of mRNA expression levels for CD59, SLC44A1, FCGR2B, TNFRSF13B, and CD320 genes. Cell populations with high expression of each gene are shown in blue, while populations with low expression of the same mRNA are shown in red. Reference datasets (GSE, GEO Series) are indicated at the top of each panel. Prognostic significance was assessed using the Cox proportional hazards model (95% confidence interval CI) and log-rank test, with p < 0.05 considered statistically significant.

Article Snippet: Antibodies included: anti-human CD45 V500 (HI30), anti-human CD19 APC-H7 (HIB19), anti-human CD59 FITC (H19), anti-human CD92 BV605 (VIM15), anti-human CD267 BV421 (1A1-K21-M22) (all from BD Biosciences, USA), anti-human CD38 PE (REA572, Miltenyi Biotec, Germany), and anti-human CD320 AlexaFluor 647 (Antibodies Online, Germany).

Techniques: Expressing

Journal: Frontiers in Medicine

Article Title: Identification of CD320, SLC44A1 and TNFRSF13B as potential novel therapeutic targets for CAR T-cell therapy in multiple myeloma

doi: 10.3389/fmed.2025.1737919

Figure Lengend Snippet: Histogram plots showing the cell surface expression of CD59, CD92, CD267, and CD320 molecules on different MM cell lines, including AMO-1, OPM-2, RPMI8226, U-266, and H929 as measured by flow cytometry analysis using fluorochrome-conjugated antibodies in triplicate experiments. Each panel represents the distribution of expression levels for the indicated marker, allowing comparison of protein abundance across the different cell lines compared to unstained control (in blue).

Article Snippet: Antibodies included: anti-human CD45 V500 (HI30), anti-human CD19 APC-H7 (HIB19), anti-human CD59 FITC (H19), anti-human CD92 BV605 (VIM15), anti-human CD267 BV421 (1A1-K21-M22) (all from BD Biosciences, USA), anti-human CD38 PE (REA572, Miltenyi Biotec, Germany), and anti-human CD320 AlexaFluor 647 (Antibodies Online, Germany).

Techniques: Expressing, Flow Cytometry, Marker, Comparison, Quantitative Proteomics, Control

Journal: Frontiers in Medicine

Article Title: Identification of CD320, SLC44A1 and TNFRSF13B as potential novel therapeutic targets for CAR T-cell therapy in multiple myeloma

doi: 10.3389/fmed.2025.1737919

Figure Lengend Snippet: Histogram plots showing the cell surface expression of CD59, CD92, CD267, and CD320 molecules on malignant plasma cells (PCs) from eight bone marrow samples of newly diagnosed, untreated MM patients as measured by flow cytometry analysis using fluorochrome-conjugated antibodies in triplicate experiments. Each panel represents the distribution of expression levels for the indicated marker across the eight MM patients samples, allowing comparison of protein abundance compared to unstained control (in blue).

Article Snippet: Antibodies included: anti-human CD45 V500 (HI30), anti-human CD19 APC-H7 (HIB19), anti-human CD59 FITC (H19), anti-human CD92 BV605 (VIM15), anti-human CD267 BV421 (1A1-K21-M22) (all from BD Biosciences, USA), anti-human CD38 PE (REA572, Miltenyi Biotec, Germany), and anti-human CD320 AlexaFluor 647 (Antibodies Online, Germany).

Techniques: Expressing, Clinical Proteomics, Flow Cytometry, Marker, Comparison, Quantitative Proteomics, Control

Journal: bioRxiv

Article Title: A Chk1-Sp1-CD59 axis of the DNA damage response impedes rituximab-mediated complement-dependent cytotoxicity

doi: 10.1101/2025.02.17.638751

Figure Lengend Snippet: (A) Schematic illustration of the multi-stage process of the phage-display antibody screen used to identify antibodies that preferentially bind to DNA-damaged cells. (B) Flow cytometry histogram of CD46 levels in MCF10A cells exposed to various doses of irradiation (0, 2, 5, 10 Gy) and stained with PE-conjugated anti-CD46 antibody or IgG-PE as isotype control. (C) Flow cytometry histogram of CD46 levels in SUDHL4 cells treated with various chemotherapy for 48H. Conditions include untreated control, IgG-PE isotype control, and treatments with 1uM etoposide (ETO), 10nM gemcitabine (GEM), 0.2mM Hydroxyurea (HU), and 2nM vincristine (VCR). (D) Representative flow cytometry histograms of CD46, CD55, and CD59 in SUDHL4 cells treated with or without etoposide (ETO). Cells were stained with PE-conjugated specific antibodies or IgG-PE as isotype control. (E) Quantification of relative integrated mean fluorescence intensities (iMFI) for CD46, CD55, and CD59 in SUDHL4 cells treated with ETO, normalized to untreated control. Statistical analysis was performed using ratio paired t-tests of IgG-corrected iMFI values. Data are presented as mean ± SD from three independent experiments. Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For mCRP neutralizing experiments, CD46 blocking antibody derived from the MC120.6 CD46 hybridoma (SFR Biosciences), CD55 antibody (BRI216) (Bio-Rad), or CD59 (YTH53.1) (Bio-rad) were added at 10µg/mL final concentration for 1 hour prior to the assay.

Techniques: Flow Cytometry, Irradiation, Staining, Control, Fluorescence

Journal: bioRxiv

Article Title: A Chk1-Sp1-CD59 axis of the DNA damage response impedes rituximab-mediated complement-dependent cytotoxicity

doi: 10.1101/2025.02.17.638751

Figure Lengend Snippet: (A) Mean cell viability of 10 DLBCL cell lines treated with 10 ug/mL rituximab (RTX) over 2 hours, measured by CTG assay. Black dots represent CDC-mediated cell death, while clear dots indicate RTX-induced direct cell death. Error bars represent SD (n≥2 biological replicates). (B) Heatmap showing log-transformed normalized transcript per million (nTPM) RNA expression values for CD20, CD46, CD55, and CD59 in DLBCL cell lines, obtained from The Human Protein Atlas, ranked by %CDC activity derived from . (C) Dose-response curve of RTX-induced cell death in SUDHL4 cells pre-treated with etoposide (ETO) for 48H, measured by CTG assay. HS, human serum; hiHS, heat-inactivated human serum. Data fitted using a four-parameter variable slope model (n=3 biological replicates). (D) Cell death induced by RTX (37 ng/mL) in SUDHL4 cells pre-treated with various chemotherapeutic agents: doxorubicin (DOX), etoposide (ETO), gemcitabine (GEM), hydroxyurea (HU), and vincristine (VCR) for 48H. Both CDC and direct cell death are shown (n=3 biological replicates). Statistical significance was calculated by 2-way ANOVA relative to respective control followed by uncorrected Fisher’s LSD. Significance of % CDC and not % apoptosis is shown. (E) Representative flow cytometry histogram of CD20 expression in SUDHL4 cells treated with or without ETO. Cells were stained with APC-conjugated anti-CD20 antibody or IgG-APC as isotype control. (F) Relative mean fluorescence intensity (MFI) of CD20 in SUDHL4 cells treated with ETO, normalized to untreated control (n=4 biological replicates). Statistical significance was calculated by ratio paired t-test of IgG-corrected MFI values. (G) Percentage of CDC in SUDHL4 cells treated with 37 ng/mL RTX including ETO-treated with blocking antibodies against CD46, CD55, or CD59 (n=3 biological replicates). Statistical significance among groups was determined using a mixed effect model, followed by post hoc Dunnett’s multiple comparisons test. Data are presented as mean ± SD. Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For mCRP neutralizing experiments, CD46 blocking antibody derived from the MC120.6 CD46 hybridoma (SFR Biosciences), CD55 antibody (BRI216) (Bio-Rad), or CD59 (YTH53.1) (Bio-rad) were added at 10µg/mL final concentration for 1 hour prior to the assay.

Techniques: CTG Assay, Transformation Assay, RNA Expression, Activity Assay, Derivative Assay, Control, Flow Cytometry, Expressing, Staining, Fluorescence, Blocking Assay

Journal: bioRxiv

Article Title: A Chk1-Sp1-CD59 axis of the DNA damage response impedes rituximab-mediated complement-dependent cytotoxicity

doi: 10.1101/2025.02.17.638751

Figure Lengend Snippet: (A) Immunoblot of Chk1 and phosphorylated Chk1 Ser345 (pChk1) in SUDHL4 cells treated with or without etoposide (ETO) for 48 hours. (B) Bar chart showing relative integrated mean fluorescence intensity (iMFI) of CD46, CD55, and CD59 in SUDHL4 cells treated with ETO alone or ETO + Chk1 inhibitor (Chk1i, Rabusertib). Statistical analysis was performed using repeated measures one-way ANOVA with Dunnett’s multiple comparisons test relative to the ETO condition. Error bars represent SD of 4 biological replicates. Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001. (C) Representative flow cytometry histograms in of CD46, CD55, and CD59 expression in SUDHL4 cells under control conditions, ETO treatment, or ETO + Chk1i treatment. Cells were stained with PE-conjugated specific antibodies or IgG-PE as isotype control. (D) Flow cytometry histograms of CD59 expression in multiple DLBCL cell lines (SUDHL6, SUDHL2, Karpas-231) under control conditions, ETO treatment, or ETO + Chk1i treatment. Cells were stained with PE-conjugated anti-CD59 antibody or IgG-PE as isotype control.

Article Snippet: For mCRP neutralizing experiments, CD46 blocking antibody derived from the MC120.6 CD46 hybridoma (SFR Biosciences), CD55 antibody (BRI216) (Bio-Rad), or CD59 (YTH53.1) (Bio-rad) were added at 10µg/mL final concentration for 1 hour prior to the assay.

Techniques: Western Blot, Fluorescence, Flow Cytometry, Expressing, Control, Staining

Journal: bioRxiv

Article Title: A Chk1-Sp1-CD59 axis of the DNA damage response impedes rituximab-mediated complement-dependent cytotoxicity

doi: 10.1101/2025.02.17.638751

Figure Lengend Snippet: (A-C) Relative mRNA expression of CD46 (A), CD55 (B), and CD59 (C) in SUDHL4 cells treated with etoposide (ETO) or ETO + Chk1 inhibitor (Chk1i, Rabusertib). (D) Schematic diagram of CD59 gene transcriptional regulators (adapted from Du et al.). (E) Sp1 and NF-κB primarily regulate transcripts T1-T4. TP53 regulates the T5 transcript but has minimal impact on overall CD59 expression. CREB primarily regulates T6-T8 transcripts and plays additional roles in CD59 transcription beyond T6-T8 regulation. An enhancer region (-500 to -1000 bp) interacts with multiple factors (CREB, NF-κB, TP53) through CBP/p300 scaffolding, facilitating coordinated regulation of CD59 expression. (E) Relative mRNA expression of CD59 exons T1-4, T5, and T6-8 in SUDHL4 cells after ETO or ETO + Chk1i treatment. (F) Percentage of complement-dependent cytotoxicity (%CDC) in SUDHL4 cells pretreated with ETO or ETO + 10 nM Mithramycin A (MitA). (G,H) Bar chart showing CD59 protein levels (MFI relative to control) (G) and relative CD59 mRNA expression (H) in SUDHL4 cells treated with control, ETO, or ETO + 10 nM MitA. (I) Immunoblot analysis of Sp1 protein and cleaved caspase-3 in SUDHL4 cells after treatment with indicated compounds. For all panels, statistical significance was calculated by ANOVA followed by post hoc Dunnett’s multiple comparisons test relative to the ETO condition. Error bars represent SD of biological replicates. Statistical significance is denoted as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For mCRP neutralizing experiments, CD46 blocking antibody derived from the MC120.6 CD46 hybridoma (SFR Biosciences), CD55 antibody (BRI216) (Bio-Rad), or CD59 (YTH53.1) (Bio-rad) were added at 10µg/mL final concentration for 1 hour prior to the assay.

Techniques: Expressing, Scaffolding, Control, Western Blot

Journal: bioRxiv

Article Title: A Chk1-Sp1-CD59 axis of the DNA damage response impedes rituximab-mediated complement-dependent cytotoxicity

doi: 10.1101/2025.02.17.638751

Figure Lengend Snippet: Schematic representation of the mechanism by which genotoxic agents antagonize rituximab-mediated complement-dependent cytotoxicity (CDC) through CD59 upregulation. Left panel: Under normal conditions, rituximab can induce CDC, resulting in membrane attack complex (MAC) formation and cell lysis. CD59 expression is driven by Sp1 and regulated by repressor complexes. Right panel: In the presence of DNA damage, genotoxic agents activate Chk1. Activated Chk1 prevents repressor complexes from associating with Sp1-bound promoter sites. Unrestricted Sp1 drives increased mCRP gene expression, particularly CD59. Elevated CD59 levels block MAC formation, inhibiting rituximab-driven CDC.

Article Snippet: For mCRP neutralizing experiments, CD46 blocking antibody derived from the MC120.6 CD46 hybridoma (SFR Biosciences), CD55 antibody (BRI216) (Bio-Rad), or CD59 (YTH53.1) (Bio-rad) were added at 10µg/mL final concentration for 1 hour prior to the assay.

Techniques: Membrane, Lysis, Expressing, Blocking Assay

Journal: Journal of Xenobiotics

Article Title: Assessment of the In Vitro Phosphatidylinositol Glycan Class A (PIG-A) Gene Mutation Assay Using Human TK6 and Mouse Hepa1c1c7 Cell Lines

doi: 10.3390/jox14030073

Figure Lengend Snippet: Mutant cell enrichment by separation with anti-CD59 microbeads and paramagnetic separator.

Article Snippet: Anti-mouse PE-conjugated CD59 antibody (Clone REA287, Lot no. 5230105732), anti-human PE-conjugated CD59 antibody (Clone REA496, Lot no. 1322120604), lyophilized anti-PE MicroBeads (Lot no. 5181221156), LS columns (Lot no. 5180927085), and a QuadroMACS separator were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Mutagenesis

Journal: Journal of Xenobiotics

Article Title: Assessment of the In Vitro Phosphatidylinositol Glycan Class A (PIG-A) Gene Mutation Assay Using Human TK6 and Mouse Hepa1c1c7 Cell Lines

doi: 10.3390/jox14030073

Figure Lengend Snippet: PIG-A gating strategy: ( a ) gating strategies for separating GPI(−) (lower left region) and GPI(+) (lower right region) of TK6 cell line; ( b ) gating strategies for separating GPI(−) (lower left region) and GPI(+) (lower right region) of Hepa1c1c7 cell line. PIG-A gene mutated cells were not stained with anti-CD59 antibodies and had no PE signal that was conjugated with antibodies.

Article Snippet: Anti-mouse PE-conjugated CD59 antibody (Clone REA287, Lot no. 5230105732), anti-human PE-conjugated CD59 antibody (Clone REA496, Lot no. 1322120604), lyophilized anti-PE MicroBeads (Lot no. 5181221156), LS columns (Lot no. 5180927085), and a QuadroMACS separator were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany).

Techniques: Staining

Journal: International Journal of Molecular Sciences

Article Title: Complement-Mediated Two-Step NETosis: Serum-Induced Complement Activation and Calcium Influx Generate NADPH Oxidase-Dependent NETs in Serum-Free Conditions

doi: 10.3390/ijms25179625

Figure Lengend Snippet: Immunofluorescence microscopy and flow cytometry show that anti-CD59 IgG-mediated classical complement activation leads to C3b and C5b-9 depositions on neutrophils. Neutrophils isolated from healthy donors were precoated with a monoclonal anti-CD59 antibodies, exposed to normal human serum, fixed and immunostained for C3b (red) and C5b-9 (green). ( A ) The confocal microscopy shows that C3b and C5b-9 were barely detectable in the control neutrophils. ( B ) The confocal microscopy shows strong staining for C3b and C5b-9 on antibody-coated neutrophils. ( C ) The quantifying fluorescence in the images shows that C3b and C5b-9 were significantly higher in the antibody-coated neutrophils than the control neutrophils. ( D ) The flow cytometry analyses also show that C3b and C5b-9 levels increased on complement-activated neutrophils compared to control neutrophils. Scale bar = 20 µm; 63× magnification. n = 3 biological replicates. ** p < 0.01, compared to their controls, based on the t-test. Data are presented as means ± SD.

Article Snippet: Neutrophils stimulated with complement were incubated with a monoclonal antibody against complement regulator, CD59 (goat anti-CD59, R&D Systems, Minneapolis, MN, USA, Cat# AF1987) reconstituted in PBS, at a concentration of 5 μg/mL for 30 min in serum-free RPMI media at 37 °C and 5% CO 2 .

Techniques: Immunofluorescence, Microscopy, Flow Cytometry, Activation Assay, Isolation, Confocal Microscopy, Control, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Complement-Mediated Two-Step NETosis: Serum-Induced Complement Activation and Calcium Influx Generate NADPH Oxidase-Dependent NETs in Serum-Free Conditions

doi: 10.3390/ijms25179625

Figure Lengend Snippet: Complement activation increases complement regulators on neutrophils. Neutrophils were precoated with anti-CD59 monoclonal antibodies and then exposed to normal human serum (NHS) to activate the complement cascade (complement stimulated). The group not treated with anti-CD59 monoclonal antibodies served as the unstimulated control (serum). The cells were then fixed, immunostained, and analyzed by flow cytometry. The expression level of all three complement regulators (CD46, CD55, and CD59) was significantly increased on complement-activated neutrophils compared to the unstimulated controls. ( A ) Representative flow cytometry tracings of an experiment; ( B ) mean fluorescence intensity (MFI) for each marker from all experiments. n = 3 biological replicates. * p < 0.05, compared to their controls, based on the paired t-test. Data are presented as means ± SD.

Article Snippet: Neutrophils stimulated with complement were incubated with a monoclonal antibody against complement regulator, CD59 (goat anti-CD59, R&D Systems, Minneapolis, MN, USA, Cat# AF1987) reconstituted in PBS, at a concentration of 5 μg/mL for 30 min in serum-free RPMI media at 37 °C and 5% CO 2 .

Techniques: Activation Assay, Bioprocessing, Control, Flow Cytometry, Expressing, Fluorescence, Marker

Journal: International Journal of Molecular Sciences

Article Title: Complement-Mediated Two-Step NETosis: Serum-Induced Complement Activation and Calcium Influx Generate NADPH Oxidase-Dependent NETs in Serum-Free Conditions

doi: 10.3390/ijms25179625

Figure Lengend Snippet: Complement activation increases the activation marker CD11b on neutrophils. Neutrophils were precoated with anti-CD59 monoclonal antibodies and then exposed to normal human serum (NHS) to activate the complement cascade (complement stimulated). The group not treated with anti-CD59 monoclonal antibodies served as the unstimulated control (serum). The cells were then fixed and immunostained for CD11b. ( A ) Immunofluorescence microscopy shows the increase in CD11b on complement-activated neutrophils compared to unstimulated controls, representative of the 3 experiments. Scale bar = 20 µm; 63× magnification. ( B ) Mean fluorescence intensity (MFI) of obtained from the flow cytometry experiments showing similar results. Complement-activated neutrophils increased the levels of CD11b, compared to unstimulated controls (serum or serum-free conditions). ( C ) The confocal microscopy shows that complement-activated or ionomycin-treated (positive control) neutrophils effectively inter-reacted with endothelial cells (incubated for 1 h and stained for DNA (DAPI, blue) and immunostained for actin (green)), compared to nonactivated control neutrophils. Arrowheads: neutrophils. Scale bar = 20 µm. Images were captured at 63× magnification. n = 3 biological replicates. * p < 0.05, compared to either control. Data are presented as means ± SD.

Article Snippet: Neutrophils stimulated with complement were incubated with a monoclonal antibody against complement regulator, CD59 (goat anti-CD59, R&D Systems, Minneapolis, MN, USA, Cat# AF1987) reconstituted in PBS, at a concentration of 5 μg/mL for 30 min in serum-free RPMI media at 37 °C and 5% CO 2 .

Techniques: Activation Assay, Marker, Bioprocessing, Control, Immunofluorescence, Microscopy, Fluorescence, Flow Cytometry, Confocal Microscopy, Positive Control, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: Complement-Mediated Two-Step NETosis: Serum-Induced Complement Activation and Calcium Influx Generate NADPH Oxidase-Dependent NETs in Serum-Free Conditions

doi: 10.3390/ijms25179625

Figure Lengend Snippet: Complement activation increases immunostaining for myeloperoxidase and citrullination of histones in neutrophils. ( A , B ) Neutrophils without ( A ) or with ( B ) precoating of anti-CD59 antibodies were incubated with serum, fixed, and immunostained for myeloperoxidase (MPO) and citrullinated histone 3 (CitH3). The confocal microscopy shows that the MPO (red) and CitH3 (green) staining levels were higher in complement-activated neutrophils compared to unstimulated controls. ( C ) Quantified fluorescence of confirmed visual observations of CitH3. ( D ) The mean fluorescence intensity (MFI) of flow cytometry analyses also shows that CitH3 levels were higher on complement-activated cells compared to control neutrophils. Scale bar = 20 µm. Images were captured at 63× magnification. n = 3 biological replicates. * p < 0.05, compared to either control. Data are presented as means ± SD.

Article Snippet: Neutrophils stimulated with complement were incubated with a monoclonal antibody against complement regulator, CD59 (goat anti-CD59, R&D Systems, Minneapolis, MN, USA, Cat# AF1987) reconstituted in PBS, at a concentration of 5 μg/mL for 30 min in serum-free RPMI media at 37 °C and 5% CO 2 .

Techniques: Activation Assay, Immunostaining, Incubation, Confocal Microscopy, Staining, Fluorescence, Flow Cytometry, Control

Journal: International Journal of Molecular Sciences

Article Title: Complement-Mediated Two-Step NETosis: Serum-Induced Complement Activation and Calcium Influx Generate NADPH Oxidase-Dependent NETs in Serum-Free Conditions

doi: 10.3390/ijms25179625

Figure Lengend Snippet: Complement activation increases intracellular calcium concentration in neutrophils. ( A ) Neutrophils were incubated with the Fluo-4 dye in the presence or absence of anti-CD59 antibodies, washed and placed in serum-free media (complement stimulated: neutrophils were precoated with anti-CD59 antibody and then exposed to NHS; unstimulated control (serum): not treated with anti-CD59 antibody; and unstimulated control (media): not treated, and no serum, only media added to the cells). Calcium levels in the cells were standardized to the initial reading (F/F0). Kinetic graphs indicate that complement activation increased intracellular calcium levels (representative of 5 experiments). ( B ) Area under the curve showing that the calcium influx into complement-activated cells is significantly higher than the control neutrophils placed in serum or serum-free media. n = 5 biological replicates. * p < 0.01, compared to controls. Data are presented as means ± SD.

Article Snippet: Neutrophils stimulated with complement were incubated with a monoclonal antibody against complement regulator, CD59 (goat anti-CD59, R&D Systems, Minneapolis, MN, USA, Cat# AF1987) reconstituted in PBS, at a concentration of 5 μg/mL for 30 min in serum-free RPMI media at 37 °C and 5% CO 2 .

Techniques: Activation Assay, Concentration Assay, Incubation, Control

Journal: International Journal of Molecular Sciences

Article Title: Complement-Mediated Two-Step NETosis: Serum-Induced Complement Activation and Calcium Influx Generate NADPH Oxidase-Dependent NETs in Serum-Free Conditions

doi: 10.3390/ijms25179625

Figure Lengend Snippet: Transitioning complement-activated neutrophils from serum to serum-free conditions induces NETosis. ( A , B ) Fluorescence microscopy showing the effect of complement activation on NETosis during the transition from serum to serum-free conditions. Neutrophils were coated with anti-CD59 monoclonal IgG, incubated in serum, fixed, and immunostained for CitH3 and MPO, with or without transitioning to serum-free conditions. Higher degrees of CitH3 and MPO colocalization and extracellular traps were detected after the transition ( B ) than before the transition ( A ). Scale bar = 20 μm; 63× magnification. Representative of n > 3 (each replicate represents an independent experiment conducted using different healthy donors for neutrophil isolation). ( C , D ) Quantitative analyses of CitH3 and DNA images, confirming the observations shown in ( A , B ). One-way ANOVA with Dunnett’s post-test. * p -value < 0.05; ** p -value < 0.01. Data are presented as means ± SD.

Article Snippet: Neutrophils stimulated with complement were incubated with a monoclonal antibody against complement regulator, CD59 (goat anti-CD59, R&D Systems, Minneapolis, MN, USA, Cat# AF1987) reconstituted in PBS, at a concentration of 5 μg/mL for 30 min in serum-free RPMI media at 37 °C and 5% CO 2 .

Techniques: Fluorescence, Microscopy, Activation Assay, Incubation, Isolation