A Method for Drug Sensitivity Activity Testing of Organoids

Organoids' vitality detection methods in drug sensitivity screening using ATP bioluminescence technology are relatively simpler and more accurate than other methods. Let me introduce the detection method to you.

 

1. Principle of ATP Bioluminescence Technology

In the organoid cell culture, the corresponding reagent is added to cause lysis of the organoids, releasing ATP. Luciferase catalyzes the conversion of the substrate luciferin, efficiently utilizing the energy of ATP to emit photons and form a light signal. The intensity of the luminescence is proportional to the amount of ATP within a certain range, and ATP is in turn proportional to the number of living cells. Therefore, it is possible to perform quantitative detection of the number of living cells in organoids.

2. Organoid ATP Bioluminescence Method

 

(1) Preparation of Organoids

Use a 24-well plate suitable for luminescence detection, and add 25 μl of a mixture of organoids and matrix gel to each well, spreading it evenly across the bottom of the well. Set up wells containing matrix gel and culture medium without organoids as negative controls. Culture the organoids according to standard organoid culture methods. If necessary, drugs can be added to treat the organoids. Additionally, if needed, a concentration gradient of organoids can be set up to determine the effectiveness of the kit later on.

 

(2) Reagent Preparation

a. Thawing: Place the ATP activity detection kit under 2~8°C or room temperature conditions to thaw;
b. Gently invert the solution several times before use to ensure uniform mixing.

 

(3) Detection Steps

a. Remove the organoid culture plate to be tested from the incubator and place it at room temperature for 30 minutes to allow the plate temperature to equilibrate to room temperature;
b. Add buffer (e.g., abs9730) equal in volume to the organoid culture and equilibrated to room temperature. For example, when using a 24-well culture plate, remove the organoid culture medium, replace it with organoid buffer, and let it sit for 3 washes, then add 25 μl of this solution to 25 μl of the organoid culture (the added volume should be consistent with the volume of the matrix gel mixture);
c. Vortex vigorously for 5 minutes to ensure complete lysis of the organoids; place at room temperature for 25 minutes to stabilize the luminescence signal before proceeding with the detection;
d. Use a multifunctional microplate reader capable of detecting chemiluminescence for chemiluminescence detection. Set the appropriate parameters according to the instrument requirements, and the detection time for each well is generally 0.25-1 second or longer, which should be adjusted according to the detection sensitivity of the instrument;
e. Calculate the relative vitality of the organoids directly from the chemiluminescence readings, or calculate the amount of ATP based on the ATP standard curve to determine the relative vitality of the organoid cells. Organoids have a good linear relationship within a density range of 0-30 organoids/well, and the ATP standard has a good linear relationship within a concentration range of 0-10,000 nM.

Note: The detection effect varies depending on the type of organoid. For some organoids with particularly high ATP content, a linear relationship may not be observed after the number of organoids reaches 100, but the chemiluminescence readings will continue to increase.

 

(4)Setting up an ATP Standard Curve (Optional):

Dilute the self-prepared ATP standard solution with PBS or organoid culture medium to appropriate concentration gradients. For initial detection, concentrations of 0, 10, 30, 100, 300, 1000, 3000, and 10,000 nM can be set, with 100 μl of standard added to each well of a 24-well plate. If necessary, the concentration range of the standards can be adjusted according to the ATP content in the organoids in subsequent experiments. If organoid culture medium is used to dilute the ATP standards, the luminescence detection should be carried out immediately after dilution, otherwise, enzymes such as ATPase in the culture medium that can consume ATP will cause a decrease in ATP content.

 

Item NO.	Product Name	Size
abs9514	Mouse Intestinal Organoid Culture Medium Kit	1kit
abs9516	Mouse Liver Organoid Culture Medium Kit	1kit
abs9515	Mouse Lung Organoid Culture Medium Kit	1kit
abs9446	Human Breast Cancer Organoid Culture Medium Kit	1kit
abs9448	Human Endometrial carcinoma Organoid Culture Medium Kit	1kit
abs9445	Human Colorectal cancer Organoid Culture Medium Kit	1kit
abs9443	Human Lung Cancer Organoid Culture Medium Kit	1kit
abs9449	Human Gastric Cancer Organoid Culture Medium Kit	1kit
abs9447	Human Pancreatic Cancer Organoid Culture Medium Kit	1kit
abs9558	Human Ovarian Cancer Organoid Culture Medium Kit	1kit
abs9544	Human Liver Organoid Culture Medium Kit	1kit
abs9546	Human Lung Organoid Culture Medium Kit	1kit
abs9549	Mouse Gastric Cancer Organoid Culture Medium Kit	1kit
abs9550	Mouse Lung Cancer Organoid Culture Medium Kit	1kit
abs9551	Mouse Breast Cancer Organoid Culture Medium Kit	1kit
abs9552	Mouse Hepatocarcinoma Organoid Culture Medium Kit	1kit
abs9553	Mouse Pancreatic Cancer Organoid Culture Medium Kit	1kit

 

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