Organoid Vitality Assay Kit 

MCE Organoid Vitality Assay Kit is a fluorescence-based assay kit specifically designed for measuring cell viability in organoids.

The detection principle is as follows: the metabolic reductases in live cells of organoids reduce the weakly fluorescent blue dye in the kit to a strongly fluorescent red product. In contrast, dead cells, due to the loss of metabolic activity, cannot reduce the dye and thus do not generate fluorescent signal. The intensity of the fluorescence signal in the system is proportional to the number of live cells, allowing the viability of the organoids to be analyzed by measuring fluorescence intensity.

Features of MCE Organoid Vitality Assay Kit

1. Ready-to-use: No need for digestion or lysis of cells and organoids.

2. Easy to operate: No washing steps are required before or after testing.

3. Cost-effective: Fluorescence-based detection principle eliminates the need for black transparent cell culture plates.

4. High sensitivity: Broad linear range, capable of detecting as few as 100 cells per well, with a minimum detection time of only 30 min.

5. Reliable data: The viability detection buffer specifically designed for organoids maintains organoid stability during activity detection, enhancing data reliability.

6. Wide applicability: The fluorescent dye is non-toxic and does not interfere with normal cellular metabolism or gene expression, enabling continuous monitoring of cell and organoid growth status.

-20℃, 2 years

Keep away from light and avoid repetitive freeze-thaw cycles.

Prepare the Organoid Viability Detection Working Solution (1×) according to the type of culture plate.

Note: For conventional cell viability assays, complete cell culture medium can be used to dilute the live cell fluorescent dye instead of the organoid viability detection solution.

Continuous Monitoring of Organoid Viability (using a 96-well plate as an example)

1. Prepare the 96-Well Plate: Remove the 96-well cell culture plate and aspirate the original complete culture medium from the wells to be tested.

Note: Aspirating the original culture medium as thoroughly as possible helps avoid further dilution of the dye by the residual medium and minimizes the impact of metabolites or culture medium components on staining quality.

2. Add Detection Working Solution: Add 100 μL of organoid viability detection working solution (1×) to each well along the well wall, and set up a well containing only dye (without organoids) as the dye negative control. Incubate in an incubator for 30 - 120 min.

Note: a. When cells or organoids are present, the color of the working solution will change from blue to pink.

b. If the cell numbers are low, the working solution may remain blue after 30 - 120 min of incubation, and the pink color may not be visibly noticeable to the naked eye. This generally does not affect the fluorescence-based detection results, and the experiment can proceed.

3. Fluorescence Detection: Use a fluorescence microplate reader (Ex/Em = 560/590 nm) to measure and record fluorescence intensity.

4. Refresh Medium: After checking, aspirate the working solution from the 96-well plate and replace it with fresh complete culture medium. Continue culturing for another 2 days.

5. Repeat Viability Detection: Repeat the organoid viability detection steps (1 - 3).

Notes: Under normal growth conditions, organoid cell viability generally increases as the culture time extends. For example, total intensity should increase by more than 20% every 2 days.

6. Analyze Relative Viability: Calculate the relative viability of the organoids based on the total fluorescence intensity (subtracting the dye-negative control).

Drug Sensitivity Assay for Organoid Viability (using a 96-well plate as an example)

Prepare different drug concentrations in advance using complete organoid culture medium.

1. Organoid Collection and Dissociation: Collect organoids and use organoid dissociation solution (MCE Cat. No.: HY-K6013) to digest them into single cells. Plate 3,000 cells per well in a 96-well plate (3–5 μL of matrix gel per well) and culture for 2 days to allow single cells to reform into organoids.

2. Prepare the 96-Well Plate for Testing: Retrieve the 96-well cell culture plate designated for testing and aspirate the complete culture medium from the wells.

Note: Thoroughly aspirating the existing culture medium helps prevent dye dilution and minimizes the effect of any residual metabolites or medium components on staining quality.

3. Add Detection Working Solution: Add 100 μL of 1× organoid viability detection working solution to each well along the well wall. Designate wells without organoids but with dye as negative dye controls. Incubate in an incubator for 30 to 120 min.

Note: a. When cells or organoids are present, the working solution color changes from blue to pink.

b. If cell numbers are low, the solution may remain blue after 30–120 min; a faint pink may not be visibly noticeable but generally does not affect fluorescence-based detection, so the experiment can proceed.

4. Fluorescence Detection: Use a fluorescence plate reader (Ex/Em = 560/590 nm) to measure and record fluorescence intensity.

5. Drug Treatment: After measurement, aspirate the working solution from the 96-well plate and apply the different drug concentrations. Set up a drug-negative control group with at least 3 replicate wells per condition. Continue culturing for 3–5 days.

6. Repeat Viability Detection: Repeat the above organoid viability detection steps (steps 2 - 4).

Note: Ensure that incubation times for measurements taken before and after drug treatment are consistent.

7. Analyze Relative Viability: Calculate the relative viability of organoids based on the total fluorescence intensity, excluding negative controls for both dye and drug.

8. Relative Viability Calculation for Drug Sensitivity Assay.

1. This product is sterile and should be handled using aseptic techniques. It is recommended to aliquot and store to avoid contamination.

2. Setting an appropriate cell density can improve detection sensitivity. For 96-well plates, it is recommended to seed between 100 to 100,000 cells per well.

3. During the assay, avoid direct sunlight exposure as much as possible. The effect of laboratory white light is negligible.

4. The incubation time for detection can be adjusted based on the cell concentration. It is recommended to use a microplate reader to measure fluorescence from the bottom of the plate to avoid interference from condensation on the plate lid and markings above the plate.

5. This product is for R&D use only, not for drug, household, or other uses.

6. For your safety and health, please wear a lab coat and disposable gloves to operate.