NB4 cells were pre-treated with OSI-906 (2.5 µM and 5 µM) for 24 h. Next day, the cells were plated in 96 well plate at the density of 500,000 cells/ml. Click-iT® OPP was added at a concentration of 20 µM while maintaining the concentration of OSI-906. Cycloheximide was added at a concentration of 50 mg/ml as a control. NB4 cells were incubated for 20 min in the incubator at 37 °C under 10% CO2. Cells were washed once with PBS, fixed in 4% paraformaldehyde and permeabilized using 0.5% Triton X-100 in PBS. According to the manufacturer’s direction click reaction was carried out with Click-iT Plus OPP Alexa Fluor 647 Protein Synthesis Assay kit (Thermo Fisher; # C10458).
Click it plus opp alexa fluor 647 protein synthesis assay kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Click-iT Plus OPP Alexa Fluor 647 Protein Synthesis Assay Kit is a tool for measuring protein synthesis in cells. It uses a chemical compound called OPP (O-propargyl-puromycin) to label newly synthesized proteins, which are then detected using Alexa Fluor 647 dye.
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Lab products found in correlation
Lsrfortessa, by BD (2 mentions)
Dmi6000 microscope, by Leica (1 mentions)
Endofit ovalbumin, by InvivoGen (1 mentions)
Cytofix cytoperm fixation permeabilization solution kit, by BD (1 mentions)
Op puro, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Orca er camera, by Hamamatsu Photonics (1 mentions)
Perm wash buffer, by BD (1 mentions)
Fixation permeabilization solution, by BD (1 mentions)
Fortessa flow cytometer, by BD (1 mentions)
Spectramax id3, by Molecular Devices (1 mentions)
12 protocols using click it plus opp alexa fluor 647 protein synthesis assay kit
1
Protein Synthesis Assay in NB4 Cells
2
Measuring Embryonic Protein Synthesis
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Embryos were incubated in no methionine DMEM (#21013024, Thermo Fisher, MA, USA) supplemented with 100 mM HPG for 30 min. followed by fixation for 30 min at 37 °C in 3.7% formaldehyde. HPG was detected using Click-iT™ HPG Alexa Fluor™ 488 (#C10428, Thermo Fisher, MA, USA). The mean signal was measured, crossed the middle of each embryo, and quantified using ImageJ software.
Embryos were incubated in M16 (Sigma–Aldrich, M7292, USA) supplemented with 100 mM OPP (O-propargyl-puromycin) for 20 min. The embryos were fixed for 30 min at 37 °C in 3.7% formaldehyde. OPP was detected using Click-iT™ Plus OPP Alexa Fluor™ 647 Protein Synthesis Assay Kit (#C10458, Thermo Fisher, MA, USA).
Embryos were incubated in M16 (Sigma–Aldrich, M7292, USA) supplemented with 100 mM OPP (O-propargyl-puromycin) for 20 min. The embryos were fixed for 30 min at 37 °C in 3.7% formaldehyde. OPP was detected using Click-iT™ Plus OPP Alexa Fluor™ 647 Protein Synthesis Assay Kit (#C10458, Thermo Fisher, MA, USA).
3
Measuring Protein Synthesis by OPP Incorporation
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Protein synthesis was determined by measuring the incorporation of O-propargyl-puromycin (OPP) into newly synthesized proteins42 (link) using the Click-it Plus OPP Alexa Fluor 647 Proteinsynthesis-Assay-Kit according to the manufacturer’s manual (Thermo Fisher). Briefly, O9-1 cells were cultured in 96-well plates until 50% confluency and a pulse of OPP was applied for 30 min. Untreated cells served as negative control, cells pre-incubated with 50 μg·mL−1 cycloheximide for 15 min defined baseline levels. After the incubation time, cells underwent fixation in 4% paraformaldehyde for 10 min before staining, image acquisition using a Leica DMI 6000 microscope, determination of the integral of the Alexa Fluor 647 intensity using FIJI43 (link) and division by the integral of the intensity of nuclear mask blue for normalization. For quantifications, three randomly chosen images per well were analyzed.
4
Quantifying Protein Synthesis In Vitro and In Vivo
5
Nascent Protein Synthesis Imaging
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Nascent protein synthesis was analyzed using Click-iT Plus OPP Alexa Fluor 647 Protein Synthesis Assay Kit (Thermo Fisher Scientific). Wandering L3 larvae were dissected in Schneider’s medium containing 5% FBS (Thermo Fisher Scientific), and incubated in 20μM OPP for 10min. After OPP incorporation, larvae were fixed in 4% paraformaldehyde for 20 min at room temperature and subsequently detected OPP by following manufacturer’s manual.
6
Measuring Protein Synthesis in P19 Cells
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P19 cells were incubated in medium supplemented with 20 μM O-propargyl-puromycin (OPP, Cayman Chemical). After 30 minutes, cells were harvested, washed with PBS, fixed with 4% PFA for 15 minutes and then permeabilized with 0.5% Triton X-100 for 15 minutes at room temperature. OPP fluorescence labeling was induced using the Click-iT Plus OPP Alexa Fluor 647 Protein Synthesis Assay Kit according to the manufacturer’s protocol (Thermo Fisher Scientific). After three PBS washes, cells were resuspended in PBS and analyzed by flow cytometry. Forward and side scatter gating was used to select a homogenous population of cells. OPP fluorescence was captured in the Cy5 channel. Two thousand events per sample were considered for analysis in three biological replicates per condition. Signal specificity was assessed by cotreatment with 100 μM cycloheximide (CHX, Sigma) or in the absence of OPP (Figure S7 A).
7
Quantifying Protein Synthesis in Thymocytes
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O-propargyl-puromycin (OPP; MedChem Source LLP) stock dissolved in 10% DMSO/PBS was diluted in PBS for intraperitoneal injection at 50 mg/kg. Mice were weighed, individually injected with OPP or vehicle, and euthanized 1 hr after injection. Thymuses were immediately harvested. Thymocytes were isolated and stained for surface antigens and viability dyes, which was followed by fixation and permeabilization according to the Click-iT Plus OPP Alexa Fluor 647 Protein Synthesis Assay Kit (ThermoFisher Scientific). Briefly, after incubation cells were subsequently fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton-X100, then incubated with the AF647 reaction cocktail. Samples were acquired using a BD LSRFortessa and analyzed using FlowJo (Becton Dickinson) as per flow cytometry methods.
8
Click-iT Protein Synthesis Assay
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For Click-iT Protein Synthesis assay, cells were first stained with surface markers to define the progenitor population, then followed by staining using a Click-iT Plus OPP Alexa Fluor 647 Protein Synthesis Assay Kit (ThermoFisher Scientific).
9
Protein Synthesis Analysis by OPP Labeling
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Protein synthesis rates were assayed by the Click-iT™ Plus OPP Alexa Fluor™ 647 Protein Synthesis Assay Kit (C10458, Thermo Fisher Scientific). Cells were pulse labelled with 10 μM O-propargyl-puromycin (OPP) for 1 h. The Click-iT™ chemistry was carried out according to manufacturer’s instructions. After OPP conjugation with Alexa Fluor™ 647, the cells were stained with 10 μM Hoechst 33342 (62249, Thermo Fisher Scientific) and 500 ng/ml Alexa Fluor™ 568 NHS Ester (SE) (A20003, Thermo Fisher Scientific) for 30 min, followed by two washings with PBS. Cells were then imaged by an Eclipse Ti microscope with the Perfect Focus System (PFS), Plan Fluor 10×/0.3 N.A. PFS dry objective lens (Nikon, Japan), and an ORCA-ER camera (Hamamatsu, Japan). Images were acquired by the NIS-Elements AR ver. 4.13.0.1 software with the WellPlate plugin.
10
In Vivo Protein Synthesis Assay
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O-propargyl-puromycin (OPP; MedChem Source LLP) stock dissolved in 10% DMSO/PBS was diluted in PBS for intraperitoneal injection at 50mg/kg. Mice were weighed, individually injected with OPP or vehicle, and euthanized one hour after injection. Thymuses were immediately harvested. Thymocytes were isolated and stained for surface antigens and viability dyes, which was followed by fixation and permeabilization according to the Click-iT Plus OPP Alexa Fluor 647 Protein Synthesis Assay Kit (ThermoFisher Scientific). Briefly, after incubation cells were subsequently fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton-X100, then incubated with the AF647 reaction cocktail. Samples were acquired using a BD LSRFortessa and analyzed using FlowJo (Becton Dickinson) as per flow cytometry methods.
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