For measurement of EdU incorporation in cells, we used Click-iT® EdU Alexa Fluor® 647 (Thermofisher C10634) according to the manufacturer’s protocol. The experiments were performed on the LSR Fortessa flow cytometer. Results were analyzed with FlowJo V10 software.
Click it edu alexa fluor 647
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Click-iT EdU Alexa Fluor 647 is a fluorescent labeling kit used for detecting newly synthesized DNA in cells. It utilizes a copper-catalyzed click reaction to incorporate a modified nucleoside, EdU (5-ethynyl-2'-deoxyuridine), into DNA during cell proliferation. The incorporated EdU is then detected using a fluorescent Alexa Fluor 647 azide dye.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
L23105, by Thermo Fisher Scientific (2 mentions)
Alex fluor 488 or 647, by Thermo Fisher Scientific (2 mentions)
Saponin, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Lsm 700 confocal microscope, by Zeiss (2 mentions)
Sc 15334, by Santa Cruz Biotechnology (1 mentions)
Sk 5100, by Vector Laboratories (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Tcs sp8 mp confocal microscope, by Leica (1 mentions)
Imagequant, by GE Healthcare (1 mentions)
Volocity, by PerkinElmer (1 mentions)
Annexin 5 apc, by Thermo Fisher Scientific (1 mentions)
Celltiter glo luminescent cell viability assay, by Promega (1 mentions)
Volocity 6, by PerkinElmer (1 mentions)
Guinea pig anti insulin, by Agilent Technologies (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Facsdiva software, by BD (1 mentions)
12 protocols using click it edu alexa fluor 647
1
Quantifying Cell Proliferation with EdU
2
EdU Labeling and Flow Cytometry
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We used the Click-iT EdU Alexa Fluor 647 (C10634; Thermo Fisher Scientific) according to the manufacturer’s protocol. 72 h after recombination, EdU was added for 4 h. Results were analyzed with FlowJo V10 software.
3
In Vivo Cell Proliferation Assessment
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Cell proliferation was assessed by EdU assay (Click-iT EdU Alexa Fluor 647, ThermoFisher, C10340) following the manufacturer’s instructions. For in vivo studies, EdU was resuspended in phosphate-buffered saline (PBS- ThermoFisher, 10010002), and 200 μl of the solution was injected intraperitoneally (50 μg per g of mouse weight) 16 hr before the sacrifice.
4
Quantifying Proliferation and Mucin Expression
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Living cells were pulsed with 5-ethynyl-2-deoxyuridine (EdU) and assayed for alkaline phosphatase (ALP). The cells then were fixed and sequentially stained for EdU incorporation (S-phase cells), mucin 2, and DNA. Cells were first pulsed with 10 μmol/L of EdU for 24 hours at 37°C, rinsed with PBS, and incubated with a red ALP substrate (SK-5100; Vector Laboratories, Burlingame, CA) in Tris buffer (0.15 mol/L, pH 8.4) for 30 minutes at 37°C. The cells were rinsed with PBS, fixed in 4% paraformaldehyde for 15 minutes, and permeabilized with 0.5% Triton X-100 in PBS for 20 minutes. Incorporated EdU was labeled with Click-iT EdU Alexa Fluor 647 (C10340; ThermoFisher). The cells were then rinsed with 0.75% glycine in PBS for 5 minutes 3 times, followed by blocking with 10% donkey serum (017-000-121; Jackson Immunoresearch, West Grove, PA) for 1 hour. The cells were incubated in rabbit mucin 2 (Muc2) antibody (1:200, sc-15334; Santa Cruz Biotechnology, Dallas, TX) at 4°C overnight, and stained with donkey anti-rabbit IgG-conjugated Alexa Fluor 488 (1:500, 711-545-152; Jackson Immunoresearch) for 45 minutes. Finally, the DNA was stained with Hoechst 33342 (2 μg/mL, B2261; Sigma-Aldrich) for 15 minutes.
5
Edu Incorporation Assay for Cardiomyocyte Characterization
6
Cell Proliferation Quantification by Click-iT EdU Assay
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Cell proliferation was measured using Click-iT® EdU Alexa Fluor 647 according to the manufacturer’s instructions (C10340, Thermo Fisher Scientific, Waltham, MA). Briefly, Click-iT® EdU Alexa Fluor 647 is a modified thymidine analogue EdU (5-ethynyl-2′-deoxyuridine, a nucleoside analog of thymidine) that is incorporated into newly synthesized DNA. The EdU is fluorescently labeled with a photostable Alexa Fluor® dye during the click reaction. Uninjured and injured cortical cells co-cultured with and without microglia for 2 DIV (days in vitro) were fixed in 3.7% formaldehyde in PBS for 15 min at RT. Fixed cells were washed twice with 1 ml of 3% BSA in PBS. Cells were permeabilized in 0.5% Triton®x-100 for 20 min at RT, washed and 1X Click-iT® EdU reaction cocktail was added for 30 min at RT. The reaction cocktail was removed, cells were washed in 3% BSA and PBS, counterstained with DAPI, mounted and imaged for analysis. Imaging was performed using IBIF Leica TCS SP8 MP Confocal Microscope at 20 × magnification. Experiments were performed in triplicate with at least 300 cells counted manually per experiment for each condition. Volocity (PerkinElmer, USA) and ImageQuant (GE Healthcare, USA) software were used for image analysis and presentation.
7
Quantifying Cell Viability, Migration, and Apoptosis
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The CellTiter-Glo Luminescent Cell Viability Assay (Promega, G7570, Madison, WI, USA) was used to measure cell survival according to the manufacturer’s instructions. A Synergy LX BioTek (Winooski, VT, USA) multi-mode plate reader was used to read luminescent signal.
Migration assays were performed as previously described56 . In experiments using JMS-053 or SU6656, cells were pre-treated with JMS-053, SU6656, or DMSO control for 2 h before plating into the upper chamber. The cells that migrated into the lower chamber were quantified by CellTiter-Glo Luminescent Cell Viability Assay.
Cell cycle was analyzed by quantifying 5′-ethynyl-2′-deoxyuridine (EdU) uptake using ClickIT EdU Alexa Fluor 647 (Thermo Fisher Scientific, C10424) according to the manufacturer’s protocol. DAPI (0.1 μg/ml) (ThermoFisher 62248) was used to stain the DNA.
Apoptosis was quantified by staining cells with Annexin V APC (ThermoFisher 88-8007-74) according to the manufacturers protocol, in the presence of DAPI (0.05 μg/ml)
Migration assays were performed as previously described56 . In experiments using JMS-053 or SU6656, cells were pre-treated with JMS-053, SU6656, or DMSO control for 2 h before plating into the upper chamber. The cells that migrated into the lower chamber were quantified by CellTiter-Glo Luminescent Cell Viability Assay.
Cell cycle was analyzed by quantifying 5′-ethynyl-2′-deoxyuridine (EdU) uptake using ClickIT EdU Alexa Fluor 647 (Thermo Fisher Scientific, C10424) according to the manufacturer’s protocol. DAPI (0.1 μg/ml) (ThermoFisher 62248) was used to stain the DNA.
Apoptosis was quantified by staining cells with Annexin V APC (ThermoFisher 88-8007-74) according to the manufacturers protocol, in the presence of DAPI (0.05 μg/ml)
8
Intestinal Stem Cell Proliferation Analysis
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Hopx-Cre-ERT2::Rosa26-LSL-tdTomato mice were injected with 2mg of tamoxifen 18 hours prior to sacrifice, and then injected with 0.3mg of 5-EdU (Thermo Fisher) per 10g of body weight 2 hours prior to sacrifice. Lgr5-eGFP-IRES-CreERT2 mice were injected with EdU 2 hours prior to sacrifice. Crypt epithelial cells were fixed and stained for EdU according to Click-iT® EdU Alexa Fluor® 647 protocol (Thermo Fisher). DNA was counterstained with DAPI. Flow cytometric analysis was performed as stated above on populations of tdTomato+ or GFP+ cells, comparing Alexa fluor 647 fluorescence to DNA content (DAPI).
9
Edu Incorporation Assay for Cardiomyocyte Characterization
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For Edu incorporation experiment (Click-iT EdU Alexa Fluor 647, Thermo Fisher, C10424), hiPSC-CM were cultured for 4 days in media containing 10μM Edu prior to dissociation. For all flow cytometry experiments, hiPSC-CM were dissociated with TrypLE 10x, washed with D-PBS, stained with the fixable dead cells stain (ThermoFisher, L23105) according to the manufacturer’s protocol, and then fixed with 4% paraformaldehyde. After washing with D-PBS, the cells were permeabilized in 1% BSA in D-PBS containing 0.1% (w/v) saponin (sigma) and incubated with primary antibodies (see Table S1 ) overnight in the permeabilization buffer. After washing, cells were incubated with secondary antibodies Alex Fluor 488 or 647 (Invitrogen) in permeabilization buffer for one hour. Edu detection was preformed according to the manufacturer’s protocol. Cell sorting/flow cytometry analysis for this project was done on instruments in the Stanford Shared FACS Facility.
10
Quantifying Beta Cell Dynamics
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Paraffin sections were prepared as described previously [23 ]. Primary antisera included guinea pig anti-insulin (Dako, Carpinteria, CA, USA) and rat anti-BrdU (Accurate Chemical, Westbury, NY, USA), followed by secondary antisera conjugated to Cy2/Cy3 (Jackson ImmunoResearch, West Grove, PA, USA) and DAPI (Molecular Probes, Eugene, OR, USA). EdU was detected using Click-iT EdU Alexa Fluor 647 (Life Technologies) according to the manufacturer’s protocol. Slides were imaged to quantify beta cell morphometry as described previously [25 (link)], using Volocity 6.1.1 (PerkinElmer, Waltham, MA, USA). BrdU-positive, EdU-positive and BrdU/EdU co-positive beta cell ratios to total beta cells were calculated, and the percentage of predicted co-positive cells was obtained by dividing the percentage of actual co-positive cells by the percentage of predicted co-positive cells, multiplied by 100%. At least 3,000 beta cells were counted per mouse. Blinding of samples was not possible given the overt phenotype.
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