rat c3 c3b  (Hycult Biotech)

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

Article Title: Role of Complement in a Rat Model of Paclitaxel-Induced Peripheral Neuropathy

doi: 10.4049/jimmunol.1701716

Figure Lengend Snippet: Design and validation of the CRISPR/Cas9 construct to introduce Indel into the exon 2 of the rat C3 gene. A. Map of the rat C3 gene; B. Validation of the C40G1 (G1) and C40G2 (G2) constructs in vitro. Activity of sgRNA C40G1 and C40G2 in cultured rat fibroblasts. A DNA fragment spanning the expected Cas9 cut site was PCR amplified and treated with T7 endonuclease I. Both C40G1 and C40G2 produced indel mutations in fibroblasts as shown by the presence of low molecular weight product in Lanes 5 and 7 (arrows). Lane 1: 100 bp ladder. Lanes 2 and 3: wild-type rat fibroblast PCR amplicon untreated (−) and treated with T7EI (+). Lanes 3 and 4: C40G1 sgRNA treated rat fibroblast PCR amplicon untreated (−) and treated with T7EI (+). Lanes 6 and 7: C40G2 sgRNA treated rat fibroblast PCR amplicon untreated (−) and treated with T7EI (+). Lanes 8 and 9: PC (positive control) PCR amplicon untreated (−) and treated with T7EI (+) shows the expected pattern.

Article Snippet: In addition, C3 deposition assays were performed by incubating sera from WT and C3 KO rats with Zymosan, followed by flow cytometric analysis of deposited C3 on the surface of Zymosan following a previously established protocol ( 23 ) using a mouse anti-human/mouse C3 mAb that cross-reacts with rat C3 (Cedarlane, clone 10C7) ( Suppl.

Techniques: Biomarker Discovery, CRISPR, Construct, Introduce, In Vitro, Activity Assay, Cell Culture, Amplification, Produced, Molecular Weight, Positive Control

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

Article Title: Role of Complement in a Rat Model of Paclitaxel-Induced Peripheral Neuropathy

doi: 10.4049/jimmunol.1701716

Figure Lengend Snippet: Identification of the founder mosaic and C3 KO rats by sequencing. The edited regions of exon 2 of the rat C3 gene were PCR amplified and sequenced; the data showed an in-frame reading in the WT rats (A and B, left panels) but not in the mosaic founder rat (A, right panel). The heterozygous (HET) rats (B, right panel) had both the WT allele (TGCT) and the KO allele (CGTCC), while the homozygous Rats (B, bottom panel) had only the KO allele (CGTCC).

Article Snippet: In addition, C3 deposition assays were performed by incubating sera from WT and C3 KO rats with Zymosan, followed by flow cytometric analysis of deposited C3 on the surface of Zymosan following a previously established protocol ( 23 ) using a mouse anti-human/mouse C3 mAb that cross-reacts with rat C3 (Cedarlane, clone 10C7) ( Suppl.

Techniques: Sequencing, Amplification

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

Article Title: Role of Complement in a Rat Model of Paclitaxel-Induced Peripheral Neuropathy

doi: 10.4049/jimmunol.1701716

Figure Lengend Snippet: Characterization of the C3 KO rats. A. Determination of serum C3 concentration. C3 levels in the sera from WT and C3 KO rats (n=5 in each group) were measured by ELISA following the manufacturer provided protocol; approximately 0.8 mg/ml C3 protein was detected in WT rats with no detectable C3 protein in the C3 KO rats. *p<0.05 B. C3 deposition assay. Serum (20%) from WT (dotted line) or C3 KO rats (solid line) were incubated with zymosan for complement activation. Then the deposited C3b/iC3b was assessed by staining the zymosan with a polyclonal anti-mouse C3 IgG (cross-react with rat C3) followed by flow cytometric analysis (shaded area, isotype control, ctrl).

Article Snippet: In addition, C3 deposition assays were performed by incubating sera from WT and C3 KO rats with Zymosan, followed by flow cytometric analysis of deposited C3 on the surface of Zymosan following a previously established protocol ( 23 ) using a mouse anti-human/mouse C3 mAb that cross-reacts with rat C3 (Cedarlane, clone 10C7) ( Suppl.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Activation Assay, Staining, Control

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

Article Title: Role of Complement in a Rat Model of Paclitaxel-Induced Peripheral Neuropathy

doi: 10.4049/jimmunol.1701716

Figure Lengend Snippet: Complement is important in paclitaxel-induced mechanical allodynia in CIPN. A. Complement is activated after paclitaxel administration. WT or C3 KO rats were i.p. injected with DMSO (Vehicle) or 1 mg/kg paclitaxel (in DMSO according to the instructions of the manufacturer, Tocris, Bristol, UK) for 4 days (day 1 to 4), and then sera were collected on day 5 when the behavioral tests confirmed the development of mechanical allodynia and probed with an anti-C3 IgG to assess complement (C3) activation. There was a significant reduction of the α band (~130KDa) of C3 and an increased α2 chain of iC3b (~40 KDa) in the paclitaxel-treated WT rats. No C3 protein was detectable in sera from the C3 KO rats. B. C3 KO rats showed increased paw withdrawal threshold (PWT). WT and C3 KO rats (n=10/group) were injected with paclitaxel for 4 days (day 1 to 4), PWT was assessed on days 0, 7 and 11.; * p<0.05.

Article Snippet: In addition, C3 deposition assays were performed by incubating sera from WT and C3 KO rats with Zymosan, followed by flow cytometric analysis of deposited C3 on the surface of Zymosan following a previously established protocol ( 23 ) using a mouse anti-human/mouse C3 mAb that cross-reacts with rat C3 (Cedarlane, clone 10C7) ( Suppl.

Techniques: Injection, Activation Assay

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

Article Title: Role of Complement in a Rat Model of Paclitaxel-Induced Peripheral Neuropathy

doi: 10.4049/jimmunol.1701716

Figure Lengend Snippet: C3 KO rats have reduced paclitaxel-induced loss of intradermal nerve fibers. WT and C3 KO rats were administered with i.p. paclitaxel for 4 consecutive days (day 1 to 4). Animals were perfused and glabrous skin of hind paws collected at day 5 when the behavioral tests confirmed the development of mechanical allodynia. A. intradermal nerve fibers in the glabrous hind paws of WT (upper panels) and C3 KO (lower panels) rats were identified by PGP 9.5 staining (red) before (left panels) and after (right panels) 4-day i.p. paclitaxel administration. DAPI (4',6-diamidino-2-phenylindole) staining (Blue) was used to for the nuclear counterstains. Pictures were taken at 20 × objectives with a fluorescent microscope. B. Quantification of surviving nerve fibers from 10 random slides taken from 3 animals/group. *p<0.05

Article Snippet: In addition, C3 deposition assays were performed by incubating sera from WT and C3 KO rats with Zymosan, followed by flow cytometric analysis of deposited C3 on the surface of Zymosan following a previously established protocol ( 23 ) using a mouse anti-human/mouse C3 mAb that cross-reacts with rat C3 (Cedarlane, clone 10C7) ( Suppl.

Techniques: Staining, Microscopy

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

Article Title: Role of Complement in a Rat Model of Paclitaxel-Induced Peripheral Neuropathy

doi: 10.4049/jimmunol.1701716

Figure Lengend Snippet: Rat neuronal cells activate complement that leads to cell damage after paclitaxel treatment. A. PC12 rat neuronal cells were cultured in the presence of 50 nM of paclitaxel or DMSO for 12, 24 and 48 hr, then incubated with 20% WT or C3 KO rat serum. Complement activation was assessed by detecting C3b/iC3 deposition on the cells using flow cytometry. Dotted lines, isotype controls; Solid lines, cells treated with paclitaxel then incubated with C3 KO serum; shaded solid lines, cells treated with paclitaxel and incubated with WT serum; shaded dot lines: cells treated with DMSO then incubated with WT serum. B. PC12 cells were cultured in the presence of 50 nM paclitaxel for 12, 24 and 48 hr, then loaded with BCECF-AM and incubated with 20% WT (WT) or C3 KO (C3 KO) serum. MAC-mediated cell damage was assessed by measuring levels of leaked BCECF in the culture supernatants.

Article Snippet: In addition, C3 deposition assays were performed by incubating sera from WT and C3 KO rats with Zymosan, followed by flow cytometric analysis of deposited C3 on the surface of Zymosan following a previously established protocol ( 23 ) using a mouse anti-human/mouse C3 mAb that cross-reacts with rat C3 (Cedarlane, clone 10C7) ( Suppl.

Techniques: Cell Culture, Incubation, Activation Assay, Flow Cytometry