Journal: Frontiers in Immunology

Article Title: TLQP-21 is a low potency partial C3aR activator on human primary macrophages

doi: 10.3389/fimmu.2023.1086673

Figure Lengend Snippet: hTLQP-2 and mTLQP-21 activate ERK signalling in CHO-K1 and CHO-C3aR cells. hTLQP-21, mTLQP-21 and plasma-derived human C3a were tested in (A) non-transfected CHO-K1 or (B) CHO cells stably expressing human C3aR. CHO cells were serum-starved overnight and then stimulated with various ligands for 10 min before being lysed. The phospho-ERK1/2 content in the lysate was measured and expressed as fold-baseline before being combined. The maximum relative pERK1/2 activity induced by each ligand is shown in (C) . Data represent mean ± S.E.M. of triplicate measurements from 3-4 independent experiments (n = 3-4). Two-way ANOVA with Dunnett’s post hoc analysis. * P < 0.05, *** P < 0.001, **** P < 0.0001. Ligand treated versus medium treated cells for each cell line.

Article Snippet: Non-transfected CHO-K1 cells or CHO cells stably expressing the human C3aR (CHO-C3aR; Product # ES-730-C, PerkinElmer, Melbourne, Australia) were maintained in Ham’s F12 medium containing 10% foetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin, with additional 400 μg/ml G418 ( In vivo Gen, San Diego, USA) added for CHO-C3aR cell culture.

Techniques: Clinical Proteomics, Derivative Assay, Transfection, Stable Transfection, Expressing, Activity Assay

Journal: Frontiers in Immunology

Article Title: TLQP-21 is a low potency partial C3aR activator on human primary macrophages

doi: 10.3389/fimmu.2023.1086673

Figure Lengend Snippet: Summary of potencies and activities of TLQP-21 tested on CHO-C3aR, HMDM and BMDM.

Article Snippet: Non-transfected CHO-K1 cells or CHO cells stably expressing the human C3aR (CHO-C3aR; Product # ES-730-C, PerkinElmer, Melbourne, Australia) were maintained in Ham’s F12 medium containing 10% foetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin, with additional 400 μg/ml G418 ( In vivo Gen, San Diego, USA) added for CHO-C3aR cell culture.

Techniques: Activity Assay

Journal: Frontiers in Immunology

Article Title: TLQP-21 is a low potency partial C3aR activator on human primary macrophages

doi: 10.3389/fimmu.2023.1086673

Figure Lengend Snippet: TLQP-21 triggers ERK signalling through C3aR in murine bone marrow-derive macrophages. BMDMs (90,000/well) from wildtype mice (A) or with C3aR knockout mice (B) , were serum-starved overnight and then stimulated with respective ligands at the indicated concentrations for 10 min. The phospho-ERK1/2 content in the cell lysate was measured and normalised to the medium only-treated levels before being combined. Data represent mean ± S.E.M. of triplicate measurements using cells from 3 mice (n = 3).

Article Snippet: Non-transfected CHO-K1 cells or CHO cells stably expressing the human C3aR (CHO-C3aR; Product # ES-730-C, PerkinElmer, Melbourne, Australia) were maintained in Ham’s F12 medium containing 10% foetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin, with additional 400 μg/ml G418 ( In vivo Gen, San Diego, USA) added for CHO-C3aR cell culture.

Techniques: Knock-Out

Journal: Experimental and Therapeutic Medicine

Article Title: The complement C3a-C3aR and C5a-C5aR pathways promote viability and inflammation of human retinal pigment epithelium cells by targeting NF-κB signaling

doi: 10.3892/etm.2022.11420

Figure Lengend Snippet: Complement C3a and C5a aggravate inflammation in HRPE cells via the NF-κB signaling pathway. HRPE cells were treated with recombinant human complement component C3a (2 µg/ml) or C5a (1 µg/ml) with or without NF-κB inhibitor PDTC (10 µM), and the release of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) PGE2 and (E) IL-10 was measured by ELISA. *** P<0.001 relative to control; ### P<0.001 relative to C3a or C5a treatment. PGE2, prostaglandin E2; HRPE, human retinal pigment epithelium.

Article Snippet: Following 24 h of treatment, the contents of various cytokines and other compounds were analyzed in HRPE cell supernatants and/or human vitreous humor were determined using the following ELISA kits in accordance with the manufacturers' protocols: Tumor Necrosis Factor-α Assay Kit (cat. no. H052-1), Interleukin-1β Assay Kit (cat. no. H002), Interleukin-6 Assay Kit (cat. no. H007-1-1) and Interleukin-10 Assay Kit (cat. no. H009-1; all from Nanjing Jiancheng Bioengineering Institute); prostaglandin E2 (PGE2) ELISA Kit (cat. no. E-EL-0034c; Elabscience Biotechnology Co., Ltd.); Human Complement Fragment 3a ELISA Kit (cat no. CSB-E08509h) and Human Complement Fragment 5a ELISA Kit (cat. no. CSB-E08512h; both from Cusabio).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Control

Journal: Experimental and Therapeutic Medicine

Article Title: The complement C3a-C3aR and C5a-C5aR pathways promote viability and inflammation of human retinal pigment epithelium cells by targeting NF-κB signaling

doi: 10.3892/etm.2022.11420

Figure Lengend Snippet: Complement C3aR and C5aR antagonist inhibit inflammation and NF-κB signaling in HRPE cells challenged with complement C3a and C5a. HRPE cells were treated with recombinant human complement component C3a (2 µg/ml) and with or without C3aR antagonist SB290157 (20 µM), and the release of (A) TNF-α, (B) IL-1β, (C) IL-6, (D) PGE2 and (E) IL-10 was determined by ELISA. HRPE cells were treated with recombinant human complement component C5a (1 µg/ml) and with or without C5aR antagonist CCX168 (2 µM), and the release of (F) TNF-α, (G) IL-1β, (H) IL-6, (I) PGE2 and (J) IL-10 was determined by ELISA. (K) The phosphorylation of NF-κB and expression of NF-κB in HRPE cells treated with recombinant human complement component C3a (2 µg/ml) and with or without C3aR antagonist SB290157 (20 µM) were determined by western blot. (L) The phosphorylation of NF-κB and expression of NF-κB in HRPE cells treated with recombinant human complement component C5a (1 µg/ml) and with or without C5aR antagonist CCX168 (2 µM) were determined by western blot. *** P<0.001 relative to control; ### P<0.001 relative to C3a or C5a treatment. p-NF-κB, phosphorylated NF-κB; C5aR, C5a receptor; HRPE, human retinal pigment epithelium; CCX, CCX168; SB, SB290157.

Article Snippet: Following 24 h of treatment, the contents of various cytokines and other compounds were analyzed in HRPE cell supernatants and/or human vitreous humor were determined using the following ELISA kits in accordance with the manufacturers' protocols: Tumor Necrosis Factor-α Assay Kit (cat. no. H052-1), Interleukin-1β Assay Kit (cat. no. H002), Interleukin-6 Assay Kit (cat. no. H007-1-1) and Interleukin-10 Assay Kit (cat. no. H009-1; all from Nanjing Jiancheng Bioengineering Institute); prostaglandin E2 (PGE2) ELISA Kit (cat. no. E-EL-0034c; Elabscience Biotechnology Co., Ltd.); Human Complement Fragment 3a ELISA Kit (cat no. CSB-E08509h) and Human Complement Fragment 5a ELISA Kit (cat. no. CSB-E08512h; both from Cusabio).

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Expressing, Western Blot, Control

Journal: Nanoscale Research Letters

Article Title: Assessment of Gold Nanoparticles-Inhibited Cytochrome P450 3A4 Activity and Molecular Mechanisms Underlying Its Cellular Toxicity in Human Hepatocellular Carcinoma Cell Line C3A

doi: 10.1186/s11671-018-2684-1

Figure Lengend Snippet: C3A viability and LC 50 values of the 40 and 80 nm a AuNP and b PC AuNP. Data represent mean ± S.D. ( n = 3). PC human plasma protein corona, ND not determined, BPEI branched polyethylenimine, LA lipoic acid, PEG polyethylene glycol, LC 50 median lethal concentration

Article Snippet: Human hepatocellular carcinoma C3A cells (ATCC ® CRL-10741TM) were purchased from ATCC ® (Manassas, VA), cultured in complete EMEM (ATCC ® , Manassas, VA) supplemented with 10% FBS, and expanded to approximately 80% confluence in T75 flask with medium changes every 4 days.

Techniques: Clinical Proteomics, Concentration Assay

Journal: Nanoscale Research Letters

Article Title: Assessment of Gold Nanoparticles-Inhibited Cytochrome P450 3A4 Activity and Molecular Mechanisms Underlying Its Cellular Toxicity in Human Hepatocellular Carcinoma Cell Line C3A

doi: 10.1186/s11671-018-2684-1

Figure Lengend Snippet: Time-dependent cellular uptake of the 40 nm a BPEI-AuNP, b LA-AuNP, and c PEG-AuNP, and the 80 nm d BPEI-AuNP, e LA-AuNP, and f PEG-AuNP in the absence and presence of PC in C3A cells up to 24 h. Data represent mean ± S.D. ( n = 3). Letters were significantly different according to Tukey’s HSD test. BPEI branched polyethylenimine, LA lipoic acid, PEG polyethylene glycol, PC human plasma protein corona, MSD minimum significant difference. * p < 0.05; ** p < 0.005; *** p < 0.0001

Article Snippet: Human hepatocellular carcinoma C3A cells (ATCC ® CRL-10741TM) were purchased from ATCC ® (Manassas, VA), cultured in complete EMEM (ATCC ® , Manassas, VA) supplemented with 10% FBS, and expanded to approximately 80% confluence in T75 flask with medium changes every 4 days.

Techniques: Clinical Proteomics

Journal: Nanoscale Research Letters

Article Title: Assessment of Gold Nanoparticles-Inhibited Cytochrome P450 3A4 Activity and Molecular Mechanisms Underlying Its Cellular Toxicity in Human Hepatocellular Carcinoma Cell Line C3A

doi: 10.1186/s11671-018-2684-1

Figure Lengend Snippet: Time- and concentration-dependent ROS/RNS production in C3A cells exposed to a the 40 nm BPEI-AuNP and b the 40 nm PEG-AuNP up to 24 h. Data represent mean ± S.D. ( n = 3). Letters were significantly different according to Tukey’s HSD test. BPEI branched polyethylenimine, LA lipoic acid, PEG polyethylene glycol, CTRL control, MSD a minimum significant difference, PCN pyocyanin (ROS inducer). ** p < 0.005; *** p < 0.0001

Article Snippet: Human hepatocellular carcinoma C3A cells (ATCC ® CRL-10741TM) were purchased from ATCC ® (Manassas, VA), cultured in complete EMEM (ATCC ® , Manassas, VA) supplemented with 10% FBS, and expanded to approximately 80% confluence in T75 flask with medium changes every 4 days.

Techniques: Concentration Assay, Control

Journal: Nanoscale Research Letters

Article Title: Assessment of Gold Nanoparticles-Inhibited Cytochrome P450 3A4 Activity and Molecular Mechanisms Underlying Its Cellular Toxicity in Human Hepatocellular Carcinoma Cell Line C3A

doi: 10.1186/s11671-018-2684-1

Figure Lengend Snippet: An inhibitory effect of AuNP on CYP3A4 activity in C3A cells exposed to the 40 and 80 nm BPEI-, LA-, and PEG-AuNP in the absence and presence of PC for 24 h. Values represent mean ± S.D. ( n = 3). BPEI branched polyethylenimine, LA lipoic acid, PEG polyethylene glycol, PC human plasma protein corona

Article Snippet: Human hepatocellular carcinoma C3A cells (ATCC ® CRL-10741TM) were purchased from ATCC ® (Manassas, VA), cultured in complete EMEM (ATCC ® , Manassas, VA) supplemented with 10% FBS, and expanded to approximately 80% confluence in T75 flask with medium changes every 4 days.

Techniques: Activity Assay, Clinical Proteomics

Journal: International Journal of Clinical Practice

Article Title: Pleural Mesothelial Cells-Induced Monocytes to the Pleural Cavity through the Effect of C3 Lytic Products in Tuberculous Pleural Effusion

doi: 10.1155/2024/5544085

Figure Lengend Snippet: Figure 2: Te concentrations of complement pyrolysis products were found higher in pleural efusion than in plasma from TPE patients. (a) Complement pyrolysis products, including C3a, C3b, C3d, C5a, and opsonin receptors (CR1 and CR3) were detected in human tuberculosis pleural biopsy samples by immunohistochemistry. (Original magnifcation, ×200) (n 4). (b) Higher levels of complement pyrolysis products were found in pleural efusion than in plasma in TPE patients (n 20). Te concentrations of complement pyrolysis products in pleural fuid and plasma from TPE patients were measured by ELISA (n 20).

Article Snippet: Monocytes isolated from TPE were incubated in the presence of medium alone or with MPT64 (20 μg/ml, Goodhere Biotechnology, Hangzhou, China), MPT64 +C3a (100 nM, 3677-C3-025, R&D Systems), MPT64+C3aRA (100 nM, SB290157, Calbiochem), or MPT64 +C3a +C3aRA.

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

Journal: International Journal of Clinical Practice

Article Title: Pleural Mesothelial Cells-Induced Monocytes to the Pleural Cavity through the Effect of C3 Lytic Products in Tuberculous Pleural Effusion

doi: 10.1155/2024/5544085

Figure Lengend Snippet: Figure 3: Monocyte migration was inhibited by antibodies that blocked CXCL12. (a) CXCL12 and CXCR4 staining by immunohisto- chemistry in human pleural biopsy (original magnifcation, ×200) (n 4). (b) Te concentration of CXCL12 in pleural fuid and plasma from TPE patients was measured by ELISA (n 20). (c) CXCL12 produced by PMCs was measured by PCR and ELISA after Mpt64 and anaphylatoxin activation. PMCs were incubated for 24 hours in control media or in media with Mpt64 (20 μg/ml) and with or without human C3a (100 nM) or C3aRA (100 nM) (n 4). (d) Coexpression of CXCL12-CXCR4 in PMCs and monocytes from TPE was detected by immunofuorescence (original magnifcation, ×400) (n 4). (e) Monocytes were seeded into the top chamber of a transwell system, and the supernatant from PMCs cultured with anti-CXCL12 antibody or PBS were placed in the bottom chamber. Te migratory index was calculated by dividing the number of monocytes that migrated in response to the supernatants from cultured PMCs by the number of monocytes that migrated in response to the control. ∗vs the MO-PBS group, ∗∗P < 0.01. #vs the MO-PMC group, ##P < 0.01 (n 4).

Article Snippet: Monocytes isolated from TPE were incubated in the presence of medium alone or with MPT64 (20 μg/ml, Goodhere Biotechnology, Hangzhou, China), MPT64 +C3a (100 nM, 3677-C3-025, R&D Systems), MPT64+C3aRA (100 nM, SB290157, Calbiochem), or MPT64 +C3a +C3aRA.

Techniques: Migration, Staining, Immunohistochemistry, Concentration Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Produced, Activation Assay, Incubation, Control, Cell Culture

Journal: Computational and structural biotechnology journal

Article Title: The complement C3a/C3aR pathway is associated with treatment resistance to gemcitabine-based neoadjuvant therapy in pancreatic cancer.

doi: 10.1016/j.csbj.2024.09.032

Figure Lengend Snippet: Fig. 8. C3aR antagonist (SB290157) attenuated the effects of C3a activation in pancreatic cancer. (A) Representative images of EdU assays showed that the treatment of SB290157 attenuated C3a-induced proliferation in Panc-1 cells. (B) The bar plot showed the percentage of EdU+ cells per field. (C) Representative images of Transwell assays showed that the treatment of SB290157 attenuated C3a-induced migration in Panc-1 cells. (D) The bar plot showed the percentage of migrated cells per field. (E) CCK-8 assay showed that the treatment of SB290157 attenuated C3a-induced gemcitabine resistance in Panc-1 cells. F-K A mouse subcutaneous tumor model of Panc-1 cells was constructed. The mice were randomly grouped and treated with vehicle, 20 mg/kg gemcitabine, 20 mg/kg SB290157, 20 mg/kg gem- citabine combined with 20 mg/kg SB29015 by intraperitoneal injection once a day. (F) Line charts of volume changes of subcutaneous tumor. (G) On the 14th day of treatment, subcutaneous tumors were separated to show tumor size. Frozen sections were prepared from tumor tissues. IF staining with antibodies to Ki-67 (in red color, (H)) and CC3 (in red color, (J)) was performed to detect proliferation and apoptosis of Panc-1 cells. And the percentage of Ki-67+ cells (I) or CC3+ cells (K) per field was shown by bar plots. * P < 0.05, * * P < 0.01, * ** P < 0.001. GEM, gemcitabine.

Article Snippet: Recombinant human complement C3a protein (MedChemExpress, MCE; HY-P7862), recombinant mouse complement C3a protein (MCE; HY-P7863) and C3aR antagonist, SB290157 (TargetMol) were used according to the manufacturer’s instructions.

Techniques: Activation Assay, Migration, CCK-8 Assay, Construct, Injection, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Complement Is Required for Microbe-Driven Induction of Th17 and Periodontitis.

doi: 10.4049/jimmunol.2200338

Figure Lengend Snippet: FIGURE 5. C3a-induced IL-6 release in human gingival epithelial cells. (A) Human immortalized gingival keratinocytes (HIGKs) were stimulated, or not, with heat-killed P. gingivalis (MOI of 10:1), C3a, or C5a and combinations thereof (at the indicated concentrations). Culture media were collected after a 24-h incubation and assayed for IL-6 by ELISA. (B) HIGKs were stimulated for 24 h with heat-killed P. gingivalis (MOI of 10:1) or Pam3Cys lipopeptide (1 mg/ml) and C3aR expression was measured by FACS. Representative histogram (left) and bar graphs for mean fluorescence intensity (MFI) of C3aR expression (right). (C) HIGKs were stimulated, or not, with C3a (500 ng/ml), Pam3Cys (1 mg/ml), or both, and IL-6 was measured in collected culture supernatants after a 24-h incubation. (D) HIGKs were stimulated for 24 h, or not, with P. gingivalis (MOI of 10:1) alone or with C3a (500 ng/ml), in the presence or absence of 10 mg/ml anti-TLR2 neutralizing Ab or isotype control (IC), which were added 2 h prior to stimulation. IL-6 release was assayed by ELISA. (E) HIGKs were pretreated with PD98059 (10 mM; MEK/ERK inhibitor), SP600125 (50mM; JNK inhibitor), SB202190 (20mM; p38 MAPK inhibitor), or SN50 (50mM, NF-kB inhibi- tor). After 1 h, Pam3Cys (1 mg/ml) was added in the cultures and C3aR expression (MFI) was determined by FACS after a 24-h incubation. (F) HIGKs were stimulated with Pam3Cys (1 mg/ml) for the indicated time lengths. Total protein was extracted and immunoblot analysis was performed with specific Abs against phosphorylated and total ERK1/2, JNK, and p38 MAPK as well as against GAPDH (loading control). (G) HIGKs were pretreated with Pam3Cys (1 mg/ml) for 4 h and then exposed to PD98059 (10 mM; MEK/ERK inhibitor), SP600125 (50mM; JNK inhibitor), SB202190 (20mM; p38 MAPK inhibitor), or SN50 (50mM, NF-kB inhibitor). After 1 h, C3a (500 ng/ml) was added in the cultures. Culture media were collected after 24 h and assayed for IL-6 by ELISA. Data are means ± SD (n 5 6 cultures per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (A, D, E, and G, one-way ANOVA and a Tukey’s test; B and C, Dunnett’s multiple comparison tests).

Article Snippet: The cells were seeded into 96-well plates at a density of 2 × 105 cells per well for 18 h and then challenged with heat-killed (65◦C, 1 h) P. gingivalis at a multiplicity of infection (MOI) of 10:1, in the presence or absence of different concentrations of recombinant human C3a or C5a (catalog no. 3677-C3-025 or 2037- C5-025/CF, R&D Systems) or the synthetic microbial lipopeptide Pam3Cys (catalog no. tlrl-pms, InvivoGen, San Diego, CA).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Fluorescence, Control, Western Blot, Comparison

Journal: Mucosal immunology

Article Title: Activation of C3a receptor is required in cigarette smoke-mediated emphysema

doi: 10.1038/mi.2014.118

Figure Lengend Snippet: Purified human C3 (100μg/ml) was cleaved with different concentrations of human ( a ) NE (10μg/ml, 1μg/ml, 0.1μg/ml) and ( b ) MMP12 (10μg/ml, 1μg/ml, 0.1μg/ml) for 4 hours at 37°C. Cleavage products were separated using 10% non-reducing Tricine gels, and detected by Western blot using anti-C3a antibody; purified C3a, NE and MMP12 were loaded as controls. ( c ) Bone marrow-derived dendritic cells (BMDCs; 5×10 4 ) and ( d ) myeloid-derived dendritic cells (MDDCs; 5×10 4 ) were suspended in media (RPMI-1640) and were placed on 48-well chemotaxis chambers for 1hr in the presence of intact or MMP12, NE cleaved C3 protein; control conditions included NE and MMP12. Transmigrating cells were detected in stained membranes visualized under microscope (20x) and reported as the average number of cells/field (n=4-6). **P<0.01, as determined by one-way ANOVA with Bonferroni's multiple comparison. Results are represented as mean±s.e.m, from 3 independent experiments.

Article Snippet: Antibodies used for western blotting and immunohistochemistry of C3 and monoclonal anti-human complement component C3a antibody were purchased from R&D Systems (Minneapolis, MN); mouse anti-human complement C3b-alpha monoclonal antibody for detection of C3b and C3c was purchased from Chemicon, Millipore (Billerica, MA); mouse anti-human C3d were purchased from AbD Serotec (Raleigh, NC).

Techniques: Purification, Western Blot, Derivative Assay, Chemotaxis Assay, Control, Staining, Microscopy, Comparison

Journal: Mucosal immunology

Article Title: Activation of C3a receptor is required in cigarette smoke-mediated emphysema

doi: 10.1038/mi.2014.118

Figure Lengend Snippet: WT and C3 deficient mice were exposed to cigarette smoke or air for 6 months. ( a ) Expression of C3ar1 mRNA in BAL cells isolated from WT or C3 −/− mice exposed to air or cigarette smoke was measured by quantitative reverse transcription PCR (qPCR). ***P<0.001, as determined by the one-way ANOVA with Bonferroni's multiple comparison. Representative ( b ) and cumulative data ( c ) measuring C3aR mean fluorescent intensity (MFI) in single lung cells gated on B220 − CD11c + population using flow cytometry. *P<0.05, as determined by the one-way ANOVA with Bonferroni's multiple comparison. ( d ) Representative photomicrograph of WT and C3 −/− mouse lung tissue exposed to six months of smoke or air immunostained for expression of C3aR (green) or nuclei (blue; DAPI). Scale bar: 20μm. Green arrows indicate C3aR + cells. ( e ) to ( h ) Mouse bone marrow-derived dendritic cells (BMDCs; 2×10 5 ) were treated with C3aR Agonist (CAS 944997-60-8; 20ng/ml) or vehicle (2% DMSO) for 48 hours. Expression level of C3aR1, Il6, Mmp9 and Mmp12 mRNA were measured using qPCR (n=4 in each group; **P<0.01, ***P<0.001, as determined by student t test. ( i ) Human CD1a + lung mDCs (2×10 5 ) were treated with purified human C3a (40ng/ml) for 24 hours or vehicle (media). Expression level of C3AR1 mRNA was measured by quantitative reverse transcription PCR (qPCR). n=3; **P<0.01, as determined by student t test. All gene expressions were normalized to 18S ribosomal RNA expression and analyzed by ΔΔCt . Results are represented as mean±s.e.m, from 3 independent experiments with 4-5 mice in each group ( a-d ).

Article Snippet: Antibodies used for western blotting and immunohistochemistry of C3 and monoclonal anti-human complement component C3a antibody were purchased from R&D Systems (Minneapolis, MN); mouse anti-human complement C3b-alpha monoclonal antibody for detection of C3b and C3c was purchased from Chemicon, Millipore (Billerica, MA); mouse anti-human C3d were purchased from AbD Serotec (Raleigh, NC).

Techniques: Expressing, Isolation, Reverse Transcription, Comparison, Flow Cytometry, Derivative Assay, Purification, RNA Expression