Cells (2 × 106) were washed twice with ice-cold PBS, suspended in 500 μl Mito buffer (10 mm HEPES, pH 6.8, 0.6 m mannitol, and 1 mm EDTA) containing 1 mm PMSF and protease inhibitor mixture (Sigma), and incubated for 15 min on ice. Cells were then disrupted by 6 cycles of freezing in liquid nitrogen and thawing at 37 °C. The crude lysate was centrifuged at 1500 × g for 10 min at 4 °C to sediment whole cells and nuclei. The supernatant was transferred to ice-cold tubes and centrifuged at 10,000 × g for 30 min at 4 °C to sediment mitochondria, and the supernatant was recovered. Protein concentration was quantified with the Bradford microplate system Gen5TM EPOCH (BioTek), and samples were analyzed by Western blot.
Epoch gen5
Manufactured by Agilent Technologies
Sourced in United States
The Epoch Gen5 is a microplate reader that provides accurate and reliable absorbance measurements for a variety of sample types and applications. It features a high-performance monochromator-based optical system and supports a wide range of microplate formats.
Automatically generated - may contain errors
Lab products found in correlation
Irdye 680lt anti mouse igg antibody, by LI COR (3 mentions)
Infrared fluorescence scanner, by LI COR (2 mentions)
Protease inhibitor mixture, by Merck Group (1 mentions)
Hsp60, by Abcam (1 mentions)
C digit blot scanner, by LI COR (1 mentions)
Hsp90, by Abcam (1 mentions)
Ez ecl system, by Sartorius (1 mentions)
Rab27a, by Santa Cruz Biotechnology (1 mentions)
Calnexin, by Abcam (1 mentions)
Tsg101, by Santa Cruz Biotechnology (1 mentions)
Ve cadherin, by Abcam (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
Immobilon p pvdf, by Merck Group (1 mentions)
Cck 8 assay, by Dojindo Laboratories (1 mentions)
Brdu labeling and detection kit, by Roche (1 mentions)
9 protocols using epoch gen5
1
Mitochondria Isolation from Cultured Cells
2
Whole Cell Protein Extraction
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Whole cell extracts and cytosolic fraction were obtained as described [6 (link)]. Protein concentration was quantified with the Bradford microplate-system Gen5TM EPOCH (BioTeK) [6 (link)].
3
Western Blot Analysis of Exosomal Markers
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Cells were detached by trypsinization and lysed in 0.2 mM HEPES (pH 7.4) containing 0.1% SDS, 1 mM Na3VO4 (phosphatase inhibitor) and protease inhibitor cocktail (10 µg/mL benzamidine, 2 µg/mL antipain, 1 µg/mL leupeptin and 1 mM PMSF). Purified exosomes were sonicated in 0.9% NaCl containing 1 mM PMSF. Protein concentration was quantified with the Bradford microplate system Gen5TM EPOCH (BioTek). Total protein extracts (30 µg/lane) were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (PVDF). Membranes were blocked with 5% skim milk in PBS/0.1% Tween (PBST) and then probed with antibodies against CD9 (m-monoclonal; Santa Cruz; 1:200), TSG101 (m-monoclonal; Santa Cruz; 1:200), ALIX (m-monoclonal; Cell Signaling; 1:500), Rab27a (m-monoclonal; Santa Cruz; 1:100), Calnexin (r-polyclonal; Abcam; 1:1000), HSP60 (r-polyclonal; Abcam; 1:20000), VE-Cadherin (m-monoclonal; Abcam; 1:1000), Lactadherin (m-monoclonal; Santa Cruz; 1:1000) and HSP90 (m-monoclonal; Abcam; 1:1000). Bound antibodies were then detected with peroxidase-labeled anti-mouse (Calbiochem 1:5000) or anti-rabbit (Calbiochem 1:3000) IgG. Blots were revealed with the EZ-ECL system (Biological Industries) on a C-DiGit Blot Scanner (LI-COR Biosciences)34 (link),36 (link).
4
Quantitative Fluorescent Protein Analysis
5
ABTS Radical Scavenging Assay for sPS
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The capability of sPS scavenging activities of 2,20-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical cation was determined with the method reported by Liu et al. with some modifications. After preparation of ABTS solution (absorbance: 0.7 ± 0.05 at 734 nm), the samples solutions (2, 1, 0.5, 0.1, and 0.05 mg/mL) were mixed with ABTS diluent. Then this mixture was kept in dark at room temperature for 6 min and the absorbance was measured at 734 nm in a microplate reader (BioTek, Epoch, Gen5). The scavenging rate (%) was used to evaluate the ABTS scavenging capacity of different sPS (Liu et al. 2016 (link)).
6
Quantitative Dual-Color Protein Immunostaining
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Proteins were quantitated (Epoch Gen5, BioTek, Winooski, VT, USA), and mixed at
1:1 with SDS sample buffer. Ten micrograms of protein were loaded on a
SDS-polyacrylamide gel electrophoresis of various concentrations depending on size
of target protein, and ran at a constant current. After transfer, the membrane
(Immobilon-P PVDF, Millipore, Billerica, MA, USA) was used for immunostaining.
Anti-α-actin mouse monoclonal antibody (AC-15, Sigma) was simultaneously
added with the antibody to the target protein. The membrane was further incubated
with IRDye 800CW-labeled secondary antibody for target protein and IRDye 680LT
anti-mouse IgG antibody (LI-COR, Lincoln, NE, USA). The membrane was
simultaneously scanned at both wave lengths on an infrared fluorescence scanner
(Odyssey, LI-COR), with target protein as green and control α-actin as
red.
1:1 with SDS sample buffer. Ten micrograms of protein were loaded on a
SDS-polyacrylamide gel electrophoresis of various concentrations depending on size
of target protein, and ran at a constant current. After transfer, the membrane
(Immobilon-P PVDF, Millipore, Billerica, MA, USA) was used for immunostaining.
Anti-α-actin mouse monoclonal antibody (AC-15, Sigma) was simultaneously
added with the antibody to the target protein. The membrane was further incubated
with IRDye 800CW-labeled secondary antibody for target protein and IRDye 680LT
anti-mouse IgG antibody (LI-COR, Lincoln, NE, USA). The membrane was
simultaneously scanned at both wave lengths on an infrared fluorescence scanner
(Odyssey, LI-COR), with target protein as green and control α-actin as
red.
7
Quantitative Protein Analysis with Infrared Scanner
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Various proteins, include crude homogenate of cortex and isolated glomeruli were quantitated (Epoch Gen5, BioTek, Winooski, VT), and mixed at 1:1 with SDS sample buffer following our published method [20 (link)]. The membrane was incubated with IRDye®800CW labeled secondary antibody for target protein and IRDye® 680LT anti-mouse IgG antibody (LI-COR, Lincoln, NE). The membrane was simultaneously scanned at both wave lengths on an infrared fluorescence scanner (Odyssey, LI-COR), with target protein as green and control α-actin as red. Each band was digitally quantitated as IOD.
8
CCK-8 Assay for Cell Viability
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Cell viability was measured using a CCK-8 assay (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. MBs cells (1×104 cells/well, 100 μl) were seeded into 96-well plates for 24 h. Fresh serum free medium containing the indicated concentrations of CCL2 or hCCL2 was added. The control ‘0’ was treated with vehicle of CCL2 or hCCL2 only (0.1% BSA in PBS). After incubation for 24 h, culture medium was replaced with drug-free medium (100 μl). Briefly, 10 μl of WST-8 dye (2-[2-methoxy-4-nitrophenyl]-3-[4-nitrophenyl]-5-[2, 4-disulfophenyl]-2H-tetrazolium, monosodium salt) was added to each well of a 96-well plate [47 (link)]. The suspension was incubated for 2 h, and the absorbance was read at 450 nm, with a reference wavelength of 650 nm, using a microplate reader (BioTek Epoch Gen5, Houston, TX, USA).
9
C2C12 Cell Proliferation Assay
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C2C12 MBs (1×104 cells/well, 100μl) were seeded into 96-well plates for 24 hours. Fresh serum free medium containing the indicated concentrations of CCL2 was added. Cell proliferation was measured by monitoring BrdU incorporation. Cells were incubated with BrdU for 3 hours, and then cell proliferation was assayed using a BrdU labeling and detection kit (Roche, Mannheim, Germany). Absorbance was measured at 450 nm, with a reference wavelength of 650 nm, using a microplate reader (BioTek Epoch Gen5).
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