GF674 EdU Cell Proliferation Assay Kit
No antibodies required; simple operation with high sensitivity.
One-click copy of information
Instruction manual- Product Description
- Composition
- Precautions
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Product Overview
EdU (5-ethynyl-2′‑deoxyuridine) is a thymidine analog that can substitute for thymine (T) and be incorporated into replicating DNA molecules. Subsequently, EdU undergoes a rapid click reaction with fluorescent dyes, resulting in fluorescent labeling of newly synthesized DNA. By detecting the fluorescent signal, cellular proliferation can be rapidly assessed. The EdU assay requires no antibodies, is simple to perform, and offers high sensitivity, representing an improved method based on the BrdU assay. This kit is suitable for analyzing both cultured cells and tissue sections.
Item number and specifications
Item number Product Name Specifications 100-121 EdU-488 Cell Proliferation Assay Kit 100T 100-122 EdU-594 Cell Proliferation Assay Kit 100T 100-123 EdU-555 Cell Proliferation Assay Kit 100T 100-124 EdU-647 Cell Proliferation Assay Kit 100T Storage conditions
This product is shipped with an ice pack and should be stored at 4°C; it remains stable for one year. Note: Hoechst 33342 (Reagent F) can be aliquoted and stored at −20°C; for short-term use, it may be kept at 4°C. Fluorescent reagents must be protected from light during handling. Shipped with an ice pack.
Self-supplied reagents
1. 10 mM PBS (pH 7.2–7.6)
2. Cell fixation solution (PBS containing 4% paraformaldehyde)
3. Permeabilization buffer (0.2% Triton X-100 in PBS)
4. Cell culture plates (48‑, 24‑, 12‑, or 6‑well plates); flow cytometry tubes (e.g., 12 × 75 mm).
Instructions for Use (Adherent Cells)
Cell culture
Seed 0.5 × 10^5 to 1 × 10^6 cells per well in a 12-well plate, ensuring normal cell growth and maintaining a confluence of no more than 80%.
EdU labeling
1. Set up one negative control group without EdU‑supplemented culture medium to enable dye background analysis of flow cytometry data.
2. Dilute the 1000× EdU solution (Reagent A) to 1× using cell culture medium, preparing an appropriate volume of 50 μM EdU–containing medium.
Note: 1) The EdU concentration is dependent on the incubation time; for short incubations (<2 h), a higher concentration (10–50 μM) is recommended, whereas for long incubations (>24 h)…
A low concentration (10 μM) is recommended;
3. Replace the cell culture medium with EdU‑containing medium, adding 0.5 mL per well, and incubate for 2 hours.
Note: 1) The optimal incubation time is cell-cycle–dependent (Table 1); for most tumor cell lines, a 2-hour incubation is suitable.
2) Reference table for EdU culture medium usage, Table 2;
Table 1. Reference for EdU Incubation Time
Cell line Human embryonic cells Human nerve cells Human cervical cancer cells Human embryonic kidney cell line Yeast cell Cell cycle 30 min 5d 20-24 hours 10–11 p.m. 3 hours Incubation time 5 minutes 1d 2 hours 2 hours 20 minutes Table 2. Reference for the Amounts of EdU Culture Medium and Staining Solution to Be Used
96-well plate 48-hole plate 24-hole plate 12-hole plate 6-hole plate EdU culture medium 100 μL 200 μL 300 μL 500 μL 1 mL Staining reaction solution 100 μL 200 μL 300 μL 500 μL 1 mL Note: Table 2 provides reference cell‑culture volumes for adherent cells; adjust the volumes accordingly for suspension cells and tissue sections.
EdU assay
a. After EdU labeling is complete, aspirate the culture medium and wash the cells once with PBS.
b. Add 0.5 mL of fixative to each well and incubate at room temperature for 10 minutes. Note: For flow cytometry, perform enzymatic digestion with trypsin prior to fixation.
c. Remove the fixative and wash the cells twice with PBS.
d. Remove the PBS, then add 0.5 mL of 0.2% Triton X-100 permeabilization buffer to each well and incubate at room temperature for 10 minutes.
e. Remove the permeabilization solution and wash the cells twice with PBS.
f. Preparation of the buffer additive solution: Dissolve one vial of buffer additive (Reagent E) in 1.3 mL of deionized water, mixing thoroughly until completely dissolved; this constitutes the buffer additive solution. After preparation, aliquot as needed and store at −20°C. If the buffer additive solution changes color to brown during use, discard it.
g. Prepare the EdU reaction solution in sequence according to Table 3:
h. Remove the PBS, add 500 μL of EdU reaction solution to each well, and incubate at room temperature in the dark for 30 minutes.
i. Remove the EdU reaction solution and wash the cells 2–3 times with PBS.
j. Nucleic acid staining: Dilute 500X Hoechst 33342 (Reagent F) with PBS to prepare a 1X staining working solution; add 0.5 mL of the working solution to each well and stain in the dark at room temperature for 5–10 minutes.
k. Remove the staining working solution and wash the cells twice with PBS.
l. Fluorescence microscopy analysis. If conditions are limited, store protected from light at 4°C in a moist environment and perform the assay within 3 days.
Table 3. Reference for the Preparation of EdU Reaction Solution
Component Number of samples on the 12-well plate 1 2 3 4 10 Reaction Buffer (Reagent B) 464 μL 928 μL 1392 μL 1856 μL 4640 μL Catalyst solution (Reagent C) 10 μL 20 μL 30 μL 40 μL 100 μL Fluorescent dye solution (Reagent D) 1 μL 2 μL 3 μL 4 μL 10 μL Buffer additive solution 25 μL 50 μL 75 μL 100 μL 250 μL Total volume 500 μL 1 mL 1.5 mL 2 mL 5 mL Note: Prepare an appropriate amount of EdU reaction solution in the specified order (prepare fresh and use within 30 minutes).
Instructions for Use (Tissue Sections)
EdU labeling in animals and processing of tissue sections
a. In vivo labeling with EdU can be performed according to relevant literature. For mice, EdU can be dissolved in PBS at a concentration of 10–200 mg/kg and administered by intraperitoneal injection, local injection into specific tissues or organs, or added to drinking water. During initial use, it is recommended to titrate the EdU concentration to determine an optimal working range.
b. After 4 hours of labeling, or at an appropriate time determined by the specific experiment, excise the required tissue and prepare frozen or paraffin sections according to standard protocols. The duration of EdU labeling may also be adjusted based on relevant literature.
c. For frozen sections:
1) A circle can be drawn around the tissue using a marking pen, and an appropriate amount of fixative should be added to the area within the circle; allow it to fix at room temperature for 15 minutes.
2) Remove the fixative and wash with PBS 2–3 times, 3 minutes each time.
3) Remove the PBS, add an appropriate volume of permeabilization buffer, and incubate at room temperature for 10–15 minutes.
4) Remove the permeabilization solution and wash twice with PBS, 3 minutes each time.
d. For paraffin sections:
1) Dewaxing: De-wax in xylene for 5–10 minutes, then replace with fresh xylene and de-wax for another 5–10 minutes. Follow with anhydrous ethanol for 2 minutes, then replace with fresh anhydrous ethanol for 2 minutes. Next, immerse in 90% ethanol for 2 minutes, 80% ethanol for 2 minutes, and 70% ethanol for 2 minutes. Finally, rinse in PBS for 5 minutes.
2) Remove the PBS, add an appropriate volume of permeabilization buffer, and incubate at room temperature for 10–15 minutes.
3) Remove the permeabilization solution and wash twice with PBS, 3 minutes each time.
EdU assay
a. Prepare the buffer additive solution: Dissolve one vial of buffer additive (Reagent E) in 1.3 mL of deionized water, mixing thoroughly until completely dissolved; this yields the buffer additive solution. After preparation, aliquot as needed and store at −20°C. If the buffer additive solution changes color to brown during use, discard it.
b. Prepare the EdU reaction solution according to the sequence listed in Table 3:
c. Remove the PBS, add an appropriate volume of EdU reaction solution to each well, and incubate in the dark at room temperature for 30 minutes.
d. Remove the EdU reaction solution and wash three times with PBS, 3–5 minutes each time.
e. Nucleic staining: Dilute 500X Hoechst 33342 (Reagent F) with PBS to prepare a 1X working stain solution. Add an appropriate volume of the working stain to each well, and incubate in the dark at room temperature for 5–10 minutes.
f. Remove the staining solution and wash the cells twice with PBS.
g. Mount the specimen using glycerol or an antifade agent.
h. Fluorescence microscopy analysis. If conditions are limited, store protected from light at 4°C in a moist environment and perform the assay within 3 days.
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Product ingredients
Component Concentration Reagent amount EdU solution (Reagent A) 1000X 100 μL Reaction Buffer (Reagent B) 1X 50 mL Catalyst solution (Reagent C) 50X 1000 μL Fluorescent dye solution (Reagent D) 500X 100 μL Buffering additive (Reagent E) powder 2 pipes Hoechst 33342 (Reagent F) 500X 100 μL Note: Fluorescence spectral data (Ex/Em): 488 Azide: 490/513 nm; 555 Azide: 550/561 nm;
594 Azide: 585/609 nm; 647A Azide: 648/664 nm.
Hoechst 33342: 346/460 nm.
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Precautions
1. Instructions on EdU concentration: This kit recommends incubating cells with 50 μM EdU for 2 hours. However, since cell type, cell density, and the rate of cell proliferation can all influence EdU incorporation during DNA replication, we advise optimizing the optimal EdU concentration in your initial experiments to achieve better results. Additionally, you may include a negative control group without EdU to facilitate analysis of the staining background.
2. During tissue section staining, it is essential to prevent the sections from drying, as drying can result in excessive background staining and compromise the experimental results.
3. Equilibrate the reagents to room temperature, briefly centrifuge, and then use.
4. For your safety and health, please wear a lab coat, a mask, and disposable gloves during the experiment.
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