Overview
In recent years, the use of 3D spheroids & tumor organoid models in high-throughput drug screening in large compound libraries and detection of single or multi-drug concentrations has demonstrated the ability to help researchers quickly screen drugs for cancer indications with strong drug sensitivity, good reactivity and excellent effect. In the detection of 2D cell drug susceptibility activity, the CCK8 method is widely used, but in the 3D tumor spheroid & tumor drug sensitivity activity, the ATP fluorescence method is more accurate than the CCK8 method.
Principles of ATP bioluminescence technology
This kit uses the bioluminescent method, which uses Firefly luciferase to catalyze the conversion of the substrate fluorescein, and efficiently uses the energy of ATP to emit photons. The luminescence signal is directly proportional to the amount of ATP present, and ATP is directly proportional to the number of cells present in organoids, which can effectively detect the activity of 3D spheroids & organoids.
Designed for multi-well plates, this kit is ideal for automated high-throughput screening (HTS), 3D spheroid & organoid proliferation, and toxicity analysis. The homogenization assay step involves adding a single reagent directly to a plate containing organoid mediumThere is no need to remove the Matrigel
Homogenization test"Dispensing-mixing-testing"The protocol is such that the 3D spheroid & organoid lysis and the resulting luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of cells in the organoid. The unique homogeneous detection protocol avoids the errors that can be introduced by ATP detection methods that require multiple steps.
Product composition
Note: The product is recommended to be used now
Features:
Simplified organoid activity assays: Homogeneous"Dispensing-mixing-testing"The protocol reduces the number of steps required for other similar tests;
Organoids are used in smaller amounts: Accurately detects the number of cells below the detection threshold of commonly used colorimetric and fluorometric methods. Reduces the number of cells required for each assay reaction;
Get results quickly: Data can be obtained 10min after adding reagent;
You can choose your own testing protocol: Can be used for many types of multi-well plate operations. Data can be recorded with a luminescence detector or CCD imaging device;
Plates can be processed continuously: The luminescence signal is stable, and the sample can be processed in batches.
How to use:
1. Preparation of organoids:
Use one that is suitable for chemiluminescence detectionWell plates(The bottom and cover are transparent, the hole wall is impermeable, recommended item number: ABS7242)., inoculated per well5ul-10ulOrganoid matrigel suspension, put in the incubator for more than half an hour, and add after coagulation100ulOrganoid medium(If using a 384-well plate, inoculate 2 per well.)5 L-4 L organoid Matrigel suspension, depending on the type of 384-well plate).and make sure that the number of cells per well is in the assayWithin 10,000 units(If a 384-well plate is used, it should be controlled within 10,000).At the same time, set up the well of the matrigel culture medium without organoids as wellNegative controlto culture organoids according to the conventional methods of organoid culture. If necessary, drug-treated organoids can be added. In addition, if necessary,It is also possible to set the concentration gradient of the organoids to determine the effectiveness of the kit later.
2. Reagent preparation:
Add 1 tube of lyophilized powder to 1 tube of 10ml buffer, mix well and protect from light and set aside.
Note: Use as soon as possible after dissolving, -80, store in the dark, valid for one month.
1) Dissolve the Organoid ATP luminescence detection reagent, if necessaryDispensingThe reagent;
2) According to the amount of 100 L per well of a 96-well plate (25 L per well of a 384-well plate), take an appropriate amount of Organoid ATP luminescence detection reagent and equilibrate to room temperature.
3. Organoid viability detection:
1) Remove the organoid culture plate and equilibrate at room temperature for 10 min (Usually it should not exceed 30min).
2) Place the organoid medium in each well of a 96-well plateRemoval, add to each well100μlOrganoid ATP luminescence detection reagent (384-well plates: 25 l per wellNote: There is no need to remove the Matrigel.
3) Room temperatureStrenuousOscillation(Microplate thermostatic shaker, recommended catalog number: ABS72034)5minOrganoids are depleted and dissolved;
4) Incubate at room temperature (about 25).minto stabilize the luminescent signal;
5) Use a chemiluminescence detection functionMultimode microplate readerChemiluminescence detection is performed. Please set the corresponding parameters according to the requirements of the instrument, and the detection time of each well is generally 025-1s or longer, depending on the detection sensitivity of the instrument, it needs to be adjusted appropriately;
6) Calculate the relative viability of organoids directly from chemiluminescence readings, or calculate the amount of ATP from the ATP standard curve to calculate the relative viability of organoids.
Note: The detection effect varies depending on the type of organoid, and for some organoids with particularly high ATP content, there may be a non-linear correlation after the number of cells reaches 50,000, but the chemiluminescence reading will continue to increase.
Frequently Asked Questions
The activity of luciferase is sensitive to temperature, so before the reactionBoth organoids and detection reagents need to be equilibrated to room temperatureThe assay is performed later. The detection reagent should be mixed well before use;
2. The detection reagent of this kit contains luciferaseRepeated freeze-thaw cycles can lead to gradual inactivation. In order to achieve a good use effect, it can be appropriate after the first dissolutionStore in aliquotsHowever, it should be noted that the containers should not be contaminated with ATP;
3. The solvent of the drug to be testedHigher levels may interfere with luciferase reactionsThus affecting the chemiluminescence signal. This can be done through the settingsOrganoid culture medium containing solvent was controlled to eliminate solvent interference;
4. The test must be suitable for organoid cultureWhite or black 96-well plates or 384-well plates. If using a plain clear 96-well plate or a 384-well plateAdjacent holes will interfere with each other;
5. Organoid seeding density, taking a 24-pass 96-well plate as an example.
Pre-passage state:24-well plates, 25 μl of matrigel organoid agglomerates per well, 500 μl of culture medium.
Passage Ratio:Organoids 1:2 passaging.
Inoculation method:24-well plates to 96-well plates.
Well inoculation requirements:5 μl of matrigel organoid agglomerate per well, 100 μl of culture medium.
Number of inoculation wells:The 24-well plate collects 3-well organoid passaging, and 6 wells should be passed according to the ratio of 1:2 passaging, with 25ul dispensing per well, and the total dispensing amount is 6*25=150ul, which is transmitted to the 96-well plate at this time, with a dispensing amount of 5ul per well, and a total number of wells of 150 5=30 wells.
Figure ICC (cholangiocarcinoma) organoid 96-well plating density map.
Detection cases
1. Organoid drug susceptibility - human hepatobiliary carcinoma (icc)
Conclusion: Human hepatocholangiocarcinoma (ICC) organoids are not sensitive to oxaliplatin, lenvatinib, larotrectinib and regorafenib. Sensitive to cisplatin, paclitaxel.
2. Organoid drug sensitivity-human lung cancer
Conclusion: Human lung cancer organoids are generally sensitive to drug1. Sensitive to drug2.
3. Organoid drug susceptibility - human pancreatic cancer
Conclusion: Human pancreatic cancer organoids are generally sensitive to irinotecan and oxaliplatin. Sensitive to paclitaxel and gemcitabine; The antimicrobial susceptibility was consistent between the two patients.
4. Antimicrobial susceptibility of organoids - human pancreatic cancer
Conclusion: Human pancreatic cancer organoids are generally sensitive to larotrectinib, oclacitinib, lenvatinib and 5-fluorouracil. The antimicrobial susceptibility was consistent between the two patients.