Polyethylenimine (PEI, MW 10000), dopamine hydrochloride and folic acid (FA) were purchased from Macklin. N-(3-Dimethylaminopropyl)-Nʹ-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and Hoechst 33258 were purchased from Sigma. The other chemical reagents used in this study were all analytically pure. Cy3-siRNA was purchased from Shanghai GenePharma Co., Ltd. LysoTracker® Green was purchased from Beyotime Biotechnology. BCA protein assay kits were purchased from Beyotime Biotechnology. Anti-ROC1, anti-Cullin1 (anti-Cul1), anti-Cullin5 (anti-Cul5), anti-ATF4, anti-P-H2AX, anti-cleaved caspase 3 and anti-Ki-67 antibodies were purchased from Abcam. CCK-8 (Cell Counting Kit-8) was purchased from MedChemExpress. An Annexin V FITC apototic kit was purchased from BD Biosciences.
Anti p h2ax
Manufactured by Abcam
Sourced in United States
Anti-P-H2AX is a primary antibody that detects phosphorylated histone H2AX, a marker of DNA double-strand breaks. It can be used in various applications such as immunofluorescence, Western blotting, and flow cytometry to measure the DNA damage response.
Automatically generated - may contain errors
Lab products found in correlation
Cell counting kit 8 cck 8, by MedChemExpress (1 mentions)
N hydroxysuccinimide, by Merck Group (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Anti ki67, by Abcam (1 mentions)
Anti cleaved caspase 3, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Folic acid, by Maclin Biochemical Technology (1 mentions)
Anti roc1, by Abcam (1 mentions)
Lysotracker green, by Beyotime (1 mentions)
Anti atf4, by Abcam (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Harmony 4, by PerkinElmer (1 mentions)
Jc 1 dye, by Thermo Fisher Scientific (1 mentions)
Thioltracker violet, by Thermo Fisher Scientific (1 mentions)
Anti cytochrome c, by Abcam (1 mentions)
3 protocols using anti p h2ax
1
Multifunctional Nanoparticles for Cancer Therapy
2
Western Blot Analysis of Phosphoproteins
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Cells were treated as described and solubilized in 20mM Tris-HCl buffer (pH7.5) containing 150 mM NaCl, 1mM EDTA, 1% NP-40, 1% sodium deoxycholate and protease inhibitor cocktail (Pierce). Samples (30 μg) were separated on 12% SDS-PAGE and then transferred to nitrocellulose. Membranes were blocked with BLOTTO and then incubated with one of the following primary antibodies for 18 hr at 4°C (Shenker et al., 1999 (link)): anti-pAkt (S473), anti pGSK3β (S9) (Cell Signaling Tech; Danvers, MA), anti-pH2AX (Abcam; Cambridge, MA) or anti-GAPDH (Santa Cruz Biotechnology). Membranes were incubated with goat anti-mouse immunoglobulin conjugated to horseradish peroxidase (Southern Biotech Technology). The Western blots were developed using chemiluminescence and analyzed by digital densitometry (Li-cor Odyssey; Li-Cor Bio; Lincoln, NE).
3
High-Content Screening for Cellular Toxicity
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The
HCS detection scheme was modified based on the method described previously.32 (link) Briefly, cells were seeded into 96-well cell
plates (PE, Cat No. 6055302, Germany) at 10 000 cells per well,
and then incubated at 37 °C for 24 h. The cells were then exposed
to different concentrations (0.2–1.0 times IC50 concentration
of each sample) of aerosol extracts for 24 h. After treatment with
aerosol extraction, cells were stained with the following probes:
ThiolTracker violet (Invitrogen, USA, Cat No.T10095) for GSH content;
DCFH-DA dye (Beyotime corp, China, Cat No. S0033M) for ROS formation;
Anti-p-H2AX (Abcam corp, USA, Cat No. ab195188) antibody for γ-H2AX
expression; Anti-c-Jun antibody for phospho-c-Jun expression; JC-1
dye (Thermo Fisher Scientific, USA, Cat No. I34361) for mitochondrial
membrane potential detection; Anti-cytochrome c (Abcam corp, USA,
Cat No. ab110325) antibody for cytochrome c release. The fluorescence
intensity was measured and automatically analyzed on a PerkinElmer
Operetta CLS high-content screening platform using Harmony 4.5 software
(PerkinElmer, Waltham, MA) and a minimum of 15 separate image fields
were selected from each well to detect the fluorescence. These results
were considered the ratio of control, which was calculated as the
change in the fluorescence of the test e-cigarette extracts compared
to the negative control.
HCS detection scheme was modified based on the method described previously.32 (link) Briefly, cells were seeded into 96-well cell
plates (PE, Cat No. 6055302, Germany) at 10 000 cells per well,
and then incubated at 37 °C for 24 h. The cells were then exposed
to different concentrations (0.2–1.0 times IC50 concentration
of each sample) of aerosol extracts for 24 h. After treatment with
aerosol extraction, cells were stained with the following probes:
ThiolTracker violet (Invitrogen, USA, Cat No.T10095) for GSH content;
DCFH-DA dye (Beyotime corp, China, Cat No. S0033M) for ROS formation;
Anti-p-H2AX (Abcam corp, USA, Cat No. ab195188) antibody for γ-H2AX
expression; Anti-c-Jun antibody for phospho-c-Jun expression; JC-1
dye (Thermo Fisher Scientific, USA, Cat No. I34361) for mitochondrial
membrane potential detection; Anti-cytochrome c (Abcam corp, USA,
Cat No. ab110325) antibody for cytochrome c release. The fluorescence
intensity was measured and automatically analyzed on a PerkinElmer
Operetta CLS high-content screening platform using Harmony 4.5 software
(PerkinElmer, Waltham, MA) and a minimum of 15 separate image fields
were selected from each well to detect the fluorescence. These results
were considered the ratio of control, which was calculated as the
change in the fluorescence of the test e-cigarette extracts compared
to the negative control.
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