FN-Click EdU Cell Proliferation Imaging Assay Kit (Green, FineTest®488)

FNCK110

50T/200T

Note: 50 T means that 50 samples can be tested with 6 well plates. EdU (10 mM) needs to be stored in aliquots for the first use(50 μL/ vial is recommended or aliquot into smaller quantities according to experimental needs).

Store at -20°C for 1 year.FineTest®488 Azide II and DAPI Reagent needs to be stored away from light.

FN-Click EdU Cell Proliferation Imaging Assay Kit (green, FineTest®488) is convenient and sensitive for proliferation detection of cell slides and smears. The results can be analyzed by fluorescence microscope.

Cell proliferation detection is widely used in the evaluation of cell activity, genotoxicity and efficacy of anti-tumor drugs. Direct detection of DNA synthesis in cells is considered to be the most accurate method to detect cell proliferation.The first widely used method to detect DNA synthesis in cells was the radionuclide incorporation method, but this method was greatly limited due to radioactive contamination and the difficulty of single-cell detection, and it was gradually replaced by the BrdU method based on antibody detection. BrdU method has many steps and requires the use of BrdU antibody, which has many influencing factors and poor stability.

EdU method is based on EdU incorporation and subsequent click reaction, without the use of antibodies, convenient operation, high detection sensitivity, is a new method to upgrade on the basis of BrdU method, will gradually replace BrdU method. EdU(5-ethynyl-2-deoxyuridine) is a thymine deoxyriboside analogue that can be incorporated into newly synthesized DNA in place of thymine deoxyriboside during DNA synthesis. On the other hand, the acetylene group on EdU can covalently react with a fluorescently labeled small molecule azide probe to form a stable triazole ring catalyzed by a monovalent copper ion, which is a very rapid reaction known as the Click reaction.Through the click reaction, the newly synthesized DNA is labeled with a corresponding fluorescent probe, so that the proliferating cells can be detected using appropriate fluorescence detection equipment.

Test results refer to the figure below:

Test:

Hela cells were treated with 10 μM EDU for 3 hours

Negative:Hela cells without EDU

1.Reagents: PBS（pH7.2~7.6); PBS (with 3% BSA) (pH7.2~7.6); Permeabilization buffer: 0.3% TritonX-100 (dissolved in PBS, pH7.2~7.6); Fixation buffer: 4% Polyformaldehyde (dissolved in PBS, pH7.2~7.6); Deionized water.

2.Instrument: fluorescence microscope.

1.Click Additive Solution: Dissolve a vial of Click Additive (220 mg) with 1.1 mL deionized water fully. Aliquot the prepared solution and store at -20ºC. (It is recommended to open a new vial of Click Additive after using one tube).

2.DAPI working solution:Add 4 μL DAPI Reagent to 96 μL PBS and mix well. Prepare the fresh solution before use.

1. Cell culture with EdU

1) The labeling concentration of EdU varies with different cell types. Cell culture medium, cell growth density, cell type and other experimental conditions may affect the labeling effect of EdU. Therefore, the labeling concentration of EdU needs to be confirmed by preliminary experiments. It is recommended to use the initial concentration of 10 μM to performe the preliminary experiment.

2)In preliminary experiments, it is recommended to set up different concentration gradients of EdU staining solution to determine the best concentration. Table 2. EdU Incubation Time for Common Cell Lines and table 3. Reference for EdU Incubation Concentration and Time in Cell Experiments can be used as reference.

Note: It is recommended to use cell sample without EdU as a negative.

2. Fixation and Permeabilization

The volume of reagents used in the following steps is suitable for 6-well plate. For other microplate, it can be adjusted appropriately according to experimental needs.

1)After incubation, discard the supernatant.

2) Add 1mL of 4% Polyformaldehyde (dissolved in PBS) to each well, incubate at RT for 15 min, and then remove the 4% Polyformaldehyde.

3)Add 1mL of PBS (with 3% BSA) to each well, and wash thoroughly for 3 times, 5 min each time.

4)Discard the supernatant, add 1mL of PBS (with 0.3% Triton X100) to each well, and incubate at RT for 20 min.

3. Labeling

This manual is based on the total reaction volume of 500 μL per well of 6-well plate. For other types of well plates, the volume of Click Reaction Solution added to each well refers to Table 1.

1) Discard the supernatant, add 1 mL of PBS (with 3% BSA) to each well, and wash thoroughly for 3 times, 5 min each time.

2)According to the number of samples, refer to the following table to prepare Click Reaction Solution.

Note: Please strictly prepare the Click Reaction Solution in accordance with the order and volume of the ingredients in the above table, otherwise it will affect the result; Click Reaction Solution should be used within 15 min after preparation.

3)Discard the supernatant, then add 500 μL of Click Reaction Solution to each well, shake gently to ensure that the Click Reaction Solution evenly covers the cells and incubate at RT for 30 min in the dark.

4)Discard the supernatant, add 1 mL of PBS (with 3% BSA) to each well and wash for 3 times, 5 min each time.

4. Nuclear staining

1) Discard the supernatant, add 500 μL of DAPI working solution to each well, and incubate at RT for 5-10 min in the dark.

2) Discard the supernatant, add 1 mL of PBS (with 3% BSA) to each well and wash for 3 times, 5 min each time.

5. Analyze

select an appropriate filter to observe the results under a fluorescence microscope.

Note: Please detect as soon as possible to avoid fluorescence quenching.

Table 1 Usage of Click Reaction Solution

Table 2 Incubation time of EdU for Common cells

Table 3 the reference of Incubation concentration and time of EdU

1.The labeling concentration of EdU should be optimized according to the cell type used. It is recommended to do a preliminary experiment to explore the optimal concentration of EdU and 10 μM EdU can be used as initial exploratory concentration.

2.Since the EdU labeling reaction is carried out in the cells and detected by fluorescence microscope, please ensure that the cells are completely fixed and permeabilized before EdU labeling. If the room temperature is too low such as in winter, it is recommended to extend the fixation time appropriately or fix it overnight at 4°C.

3.Aliquot the Click Additive Solution and store at -20ºC. If white substance is precipitated before use, please turn it upside down several times and use it only after it has completely dissolved. If the color of the Click Additive Solution turns brown, indicates that the reagent has expired, please discard it.

4.Copper ions will affect the fluorescence of GFP, RFP, mCherry and other fluorescent proteins, so this kit is not suitable for cells with GFP, RFP, mCherry and other fluorescence.

5.For your safety and health, please wear a lab coat and disposable gloves.

6.This kit is for scientific research only.