C300 digital chemiluminescent imager

Manufactured by Azure Biosystems

The C300 digital chemiluminescent imager is a laboratory instrument designed for the detection and analysis of chemiluminescent signals. It utilizes a high-sensitivity CCD camera to capture and digitize chemiluminescent images.

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C300 imager (5 citations)

4 protocols using c300 digital chemiluminescent imager

Western Blot Analysis of Extracellular Vesicles and Brain Tissue

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For EV samples, after being lysed with TENT buffer, the equivalent amount of protein was loaded on 4–20% SDS-PAGE gels (Bio-Rad). For mouse brain tissue samples, homogenates of hippocampus from each experimental group and an equal proportion of corresponding homogenates, S1, S1p and P2, were loaded on 10% SDS-PAGE gels (Bio-Rad) and electrotransferred to 0.45 μm nitrocellulose membranes (Bio-Rad). For dot blotting, an equal volume of EV samples were dotted onto 0.45 μm nitrocellulose membranes (Bio-Rad) and washed twice with TBS buffer. The membranes were then blocked in freshly prepared 5% BSA diluted in TBS before being immunoblotted with specific primary antibodies (Supplementary Table 1). The membrane was further incubated with HRP-labelled secondary antibodies (Santa Cruz Biotech) and scanned using the C300 digital chemiluminescent imager (Azure Biosystems). The band densities were digitally measured using ImageJ (NIH).

Ruan Z., Pathak D., Venkatesan Kalavai S., Yoshii-Kitahara A., Muraoka S., Bhatt N., Takamatsu-Yukawa K., Hu J., Wang Y., Hersh S., Ericsson M., Gorantla S., Gendelman H.E., Kayed R., Ikezu S., Luebke J.I, & Ikezu T. (2020). Alzheimer’s disease brain-derived extracellular vesicles spread tau pathology in interneurons. Brain, 144(1), 288-309.

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EV Protein Profiling by Immunoblotting

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For EV samples, after lysis with TENT buffer (50mM Tris-HCl, 2mM EDTA, 150mM NaCl, 1% Triton-X 100, pH7.5), an equivalent amount of protein was loaded on 4–20% sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS–PAGE) gels (#4561093, Bio-Rad) and electro-transferred to 0.45 μm nitrocellulose membranes (#162-0090, Bio-Rad). The membranes were blocked in freshly prepared 5% skim milk diluted in TBS before being immunoblotted with specific primary antibodies (anti-rabbit CD63, Biorbyt, # orb229829), anti-Tsg101 (1:100, clone Y16J, Santa Cruz Biotechnology), tdTomato, Thermo Fisher, TA150128, mouse mAb, anti-early endosome antigen A1 (EEA1, 1:100, clone G-4, Santa Cruz Biotechnology), anti-Rab7 (1:1,000, clone D95F2, Cell Signaling Technology), anti-calregulin (1:200, clone F-4, Santa Cruz Biotechnology), anti-cytochrome C antibody (1:100, clone 7H8, Santa Cruz Biotechnology), anti-GM130 (1:100, clone NN2C10/1, Santa Cruz Biotechnology), anti-Histone H2A.Z (1:400, 2718, Cell Signaling Technology), anti-LC3 a/b antibody (1:1,000, 4108S, Cell Signaling Technology), anti-β-actin (1:2,000, A1978, Sigma-Aldrich). The membrane was further incubated with HRP-labeled secondary antibodies (Cell Signaling Technology) and scanned using a C300 digital chemiluminescent imager (Azure Biosystems). The band densities were digitally measured using ImageJ software (NIH).

Ruan Z., Takamatsu-Yukawa K., Wang Y., Ushman M.L., Labadorf A.T., Ericsson M., Ikezu S, & Ikezu T. (2022). Functional genome-wide short hairpin RNA library screening identifies key molecules for extracellular vesicle secretion from microglia. Cell reports, 39(6), 110791.

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EV Protein Profiling by Immunoblotting

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Quantitative Western and Dot Blotting

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For western blotting, homogenates (Ho) of hippocampus from each experimental group and an equal proportion of corresponding Ho, S1, S1p and P3, were loaded on 10% SDS-PAGE gels ( Bio-Rad) and electro-transferred to 0.45-μm nitrocellulose membranes (Bio-Rad). For dot blotting, an equal volume of EVs sample were dotted onto 0.45-μm nitrocellulose membranes (Bio-Rad) and washed twice with TBS buffer. The nitrocellulose membranes were then blocked in freshly prepared 5% skim milk diluted in TBS before being immunoblotted with specific primary antibodies (Supplementary Table 3). The membrane was further incubated with HRPlabeled secondary antibodies and scanned using C300 digital chemiluminescent imager (Azure Biosystems). The optical densities were measured using Image J software.

Ruan Z., Pathak D., Kalavai S.V., Yoshii-Kitahara A., Muraoka S., Takamatsu-Yukawa K., Bhatt N., Hu J., Wang Y., Hersh S., Gorantla S., Kayed R., Gendelman H.E., Ikezu S., Luebke J.I., & Ikezu T. (2020). Alzheimer’s disease brain-derived tau-containing extracellular vesicles: Pathobiology and GABAergic neuronal transmission.

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