Cell lysates (100 000 cell equivalents per well) were probed with antibodies to GAPDH (R&D Systems), β-actin (Sigma-Aldrich), and ACE2 (Novus Biologicals). The appropriate secondary horseradish peroxidase antibodies were used for imaging with an iBright 1500 system (Invitrogen), and ImageJ (Version 1.49 23) software was used for quantification. Cell surface expression was measured by means of flow cytometry using ACE2–phycoerythrin (PE) (Novus Biologicals), as recommended by the manufacturer. Primary cell populations were incubated with CD19-PacificBlue (BioLegend), CD3–fluorescein isothiocyanate (FITC), CD4-FITC, CD8-FITC, CD14– allophycocyanin (APC), and CD56-AlexaFluor700 (all BD Biosciences). Primary cells were treated with human immunoglobulin G (10 µg/1 million cells) to block Fc receptors on B cells. Data were acquired on an LSR II flow cytometer using a single-stained AbC bead kit (ThermoFisher) for compensation [17 (link)]. Viable cells were gated based on forward and side scatter, doublets were excluded, and “fluorescence minus 1” controls were assessed. PE quantitation beads (BD Quantibrite) were analyzed, according to the manufacturer’s instructions, to quantify ACE2 molecules per cell. At least 10 000 total events were collected in each experiment and the FlowJo program (Tree Star) was used for data analysis.
Ibright 1500 system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The IBright 1500 system is a compact and versatile imaging platform designed for a wide range of microscopy applications. It offers high-quality imaging capabilities, enabling researchers to capture and analyze images with precision and efficiency.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (2 mentions)
Np 40 buffer, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Hrp conjugated anti rabbit secondary antibody, by Thermo Fisher Scientific (2 mentions)
Cd8 fitc, by BD (1 mentions)
Cd4 fitc, by BD (1 mentions)
Gapdh, by R&D Systems (1 mentions)
Cd19 pacific blue, by BioLegend (1 mentions)
β actin, by Merck Group (1 mentions)
Cd56 alexafluor700, by BD (1 mentions)
Mouse anti β actin, by Proteintech (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Mouse anti tyrosine hydroxylase, by Merck Group (1 mentions)
Alkaline phosphatase, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Radio immunoprecipitation assay lysis buffer, by Biosharp (1 mentions)
Hsp70, by ABclonal (1 mentions)
Phospho akt, by ABclonal (1 mentions)
7 protocols using ibright 1500 system
1
ACE2 Expression Quantification in Primary Cells
2
MPTP-Induced Dopaminergic Neuron Loss
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The mice were sacrificed on day 1 after the last administration of MPTP. The brain tissues of the striatum were isolated and stored at −80°C. The brain tissues were homogenized in 1X RIPA lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and phosphatase inhibitor cocktail (Roche, Basel, Switzerland). The lysate was centrifuged at 4°C and 12,000 g for 15 min. Equal amounts of protein from all groups were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% gel. The resolved proteins were transferred to a 0.45-μm polyvinylidene fluoride (PVDF) membrane. The membrane was blocked using 5% (w/v) skimmed milk to inhibit non-specific binding. Next, the membrane was incubated overnight at 4°C with the following primary antibodies: mouse anti-tyrosine hydroxylase (TH) (Sigma, 1:000) and mouse anti-β-actin (Proteintech, 1:1000) antibodies. The membrane was then incubated with the HRP-conjugated goat anti-mouse (CST, United States 1:1000) secondary antibodies for 1 h. The proteins were visualized using the enhanced chemiluminescence (ECL) test kit and Invitrogen iBright 1,500 system.
3
Quantitative OspC Immunoassay Development
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Recombinant OspC types A, B, and K (Table 1 ) at 0.5 μg/μL, were 5-fold serially diluted in PBS, and 2 μL of each dilution was spotted on a dry nitrocellulose membrane. The spots were allowed to air dry for 1 h and then were blocked with 5% milk in 1× Tris-buffered saline with Tween 20 (TBS-T) for 18 h and incubated with 0.1 μg/mL chimeric B5 IgG1 in 5% milk in 1× TBS-T at room temperature for 1 h. The membrane was then washed twice with 1× TBS-T, incubated with a 1:10,000 dilution of goat anti-human IgG (H+L)-horseradish peroxidase (HRP; Invitrogen), and washed twice more before detection with enhanced chemiluminescence (ECL; ECL Plus Western blotting substrate; Pierce, Thermo Fisher, Waltham, MA). Images were acquired and analyzed using an iBright 1500 system (Invitrogen).
4
Protein Extraction and Detection
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Five oocytes (15 mg) or 10 mg fresh plant leaf tissue was homogenized in 100 µl of 2× SDS sample buffer. After 10 min boiling, cell lysates were briefly centrifuged and 10 µl was loaded to each lane of an SDS–PAGE gel. After separation, proteins were blotted onto a PVDF membrane. AvrE, β-actin, DspE or PR1 was detected by anti-AvrE20 (link) (1:5,000 dilution), anti-β-actin [HRP] (GenScript; 1:5,000), anti-DspE50 (link) (1:5,000), anti-PR1 antibody (a gift from Xinnian Dong; 1:5,000), respectively, on an Invitrogen iBright 1500 system. The secondary antibodies anti-rabbit IgG (whole molecule)–alkaline phosphatase (Sigma) or anti-rabbit IgG (whole molecule)–HRP antibody (Sigma) were used with 1:10,000 or 1:5,000 dilution, respectively.
5
Exosome Protein Isolation and Immunoblotting
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Protein was extracted from an exosome using a Total Exosome Protein Isolation Kit (ThermoFisher). The cultured cells were lysed in Radio Immunoprecipitation Assay Lysis Buffer (Biosharp). The protein sample was separated using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE, Bio‐Rad) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The following antibodies were used as primary antibodies: Alix (ab186429, Abcam), TSG101 (A1692; Abclonal), CD63 (A19023, Abclonal), HSP70 (A12948; Abclonal), phospho‐PI3K (AP0854, Abclonal), PI3K (A11526, Abclonal), phospho‐Akt (AP0637, Abclonal), Akt (A11016, Abclonal), phospho‐FoxO1 (AP0172, Abclonal), FoxO1 (A2934, Abclonal), phospho‐FoxO3a (AP0684, Abclonal), FoxO3a (A0102, Abclonal), and β‐actin (AC026, Abclonal). Secondary antibodies were horseradish peroxidase (HRP) goat anti‐rabbit IgG (AS014, Abclonal) and goat anti‐mouse antibodies IgG (AS003, Abclonal). Visualization and analysis were performed using the iBright1500 system (Thermo).
6
Histone Acetylation Regulation by TSA
7
Histone Deacetylase Inhibitor TSA Regulates TP53 Acetylation
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Human HEK293 and HCT116 cells were cultured in EMEM (ATCC, 30‐2003) and DMEM Gibco # 11995‐065) medium, respectively. Cells were treated with 4 μM histone deacetylase inhibitor Trichostatin A (TSA) (Sigma, T8552) or DMSO for 24 hours, then collected and lysed in NP40 buffer (Fisher, FNN0021) for Western blot analysis. Zebrafish wild‐type 24 hpf embryos were treated with 0.5 μM TSA for 24 and 48 hours. A 20 μg of total protein extracted from cell and zebrafish embryo samples was run on a 10% SDS‐PAGE gel, blotted to PVDF membrane (Bio Rad, 1620176), and incubated with rabbit monoclonal anti‐TP53‐acetylated‐K370 (abcam, ab183544) at 1:500, rabbit polyclonal anti‐β actin (Cell Signaling, 4967) at 1:2000, rabbit polyclonal anti‐RBBP4 (Bethyl A301‐206A‐T, RRID: AB_890631) 1:2000, and HRP‐conjugated anti‐rabbit secondary antibody (Invitrogen, 31 460) at 1:5000. Blots were developed with SuperSignal West Dura Extended Duration Substrate (ThermoScientific, 34075) and imaged on a ThermoFisher iBright 1500 system.
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