Organoid In Situ Live-Dead Staining Experiment Guide

Overview

 

During the cultivation process of organoids, if we want to understand the proliferation and apoptosis status of organoids, we can observe through fluorescence staining of live and dead cells and take photos with a fluorescence microscope.

 

Calcein-AM is a cell staining reagent for fluorescently labeling live cells, emitting green fluorescence (Ex=490nm, Em=515nm). It introduces an acetoxymethyl (AM) group to the traditional Calcein (calcium green), increasing its hydrophobicity and allowing it to easily penetrate the cell membrane of live cells. Once inside the cell, Calcein-AM (which is non-fluorescent) is cleaved by cellular esterases to form a membrane-impermeable polar molecule, Calcein, which is retained within the cell and emits a strong green fluorescence. Compared to other similar reagents (such as BCECF-AM and CFDA), Calcein-AM has a very low cytotoxicity and is the most suitable fluorescent probe for staining live cells without inhibiting any cellular functions, such as proliferation and chemotaxis of lymphocytes.  

Since dead cells lack esterases, Calcein-AM is only used for the cell viability test and short-term marking of live cells. Therefore, Calcein-AM is often used in conjunction with dead cell fluorescence probes such as Propidium Iodide (PI) for the simultaneous fluorescence double staining of live and dead cells. Propidium Iodide (PI) cannot pass through the cell membrane of live cells, but can only pass through the disordered areas of the dead cell membrane to reach the nucleus and intercalate into the DNA double helix of the cell, thereby producing red fluorescence (Ex=535nm, Em=617nm), so PI only stains dead cells. Since both Calcein and PI-DNA can be excited by 490nm, live and dead cells can be observed simultaneously with a fluorescence microscope. When excited with 545nm, only dead cells can be observed.

 

Principle

 

The working principle of this kit is based on the double staining with Calcein-AM and PI to mark live and dead cells, thereby analyzing the levels of live and dead cells. According to our company's optimized experimental system, for example, using a 24-well plate with 25uL organoid matrix gel coagulates per well, add 500uL of staining working solution per well for staining.

 

Calcein-AM/PI Double Stain Kit(abs50056)

Component	Name	Size	Storage Conditions
A	Calcein-AM Solution(2mM)	50μL	Store at -20°C in the dark and dry conditions
B	PI Solution(1.5mM）	150μL	Store at -20°C in the dark and dry conditions
C	10×Assay Buffer	50mL	Store at -20°C; for frequent use, it can be kept at 4°C

 

Experimental Procedure

 

I. Preparation of Working Solutions:

1. Preparation of 1×Assay Buffer: Remove the 10×Assay Buffer from the cold storage and, under sterile conditions, take an appropriate amount for a single use and dilute it 10 times with deionized water (dH2O) to obtain 1×Assay Buffer.

2. Preparation of 1×Staining Working Solution:

(1) First, allow the Calcein-AM solution (2mM) and PI solution (1.5mM) stored at low temperature to return to room temperature for 20-30 minutes. Note: For the first use, the mother solution can be aliquoted to reduce the number of freeze-thaw cycles.

(2) Add 5μL of Calcein-AM solution (2mM) and 15μL of PI solution (1.5mM) to 5mL of 1×Assay Buffer and mix well. At this point, the working concentration of Calcein-AM is 2μM, and the working concentration of PI is 4.5μM.

Note: Since the optimal staining conditions vary for different types of organs, it is recommended to perform gradient experiments for the first time to determine the optimal concentrations of Calcein-AM and PI. The principle of gradient screening is to use the lowest probe concentration to achieve the best fluorescence results. The following methods can be used to optimize the best working concentrations for both fluorescent dyes.

 

a. Incubate organoids with 0.1% saponin or 0.1-0.5% digitonin for 1 hour, or with 70% ethanol for 1 hour, to prepare dead organoids;

b. Stain dead organoids with a PI solution at 0.1-10μM to obtain the optimal working concentration that stains only the cell nuclei without staining the cytoplasm;

c. Stain dead organoids with a Calcein-AM solution at 0.1-10μM to obtain the optimal working concentration that does not stain the cytoplasm. Then use this concentration to stain live cells to observe whether live cells can be stained.

 

II. Staining Steps:

 

Using a 24-well plate with 25uL of organoid matrix gel coagulates per well as an example, simply discard the organoid culture medium during detection, retain 25μL of organoid matrix gel coagulates in the well, and add the staining working solution.

1. Aspirate the organoid culture medium, add PBS and rinse 2-3 times to fully remove residual esterase activity, then discard the PBS;

2. Add 500μL of staining working solution per well, mix well, and incubate at 37℃ for 1 hour;

3. Detect live cells (yellow-green fluorescence) and dead cells (red fluorescence) simultaneously under a fluorescence microscope using an excitation filter of 490±10nm. Additionally, using an emission filter of 545nm will only allow observation of dead cells. Detection can also be performed directly under a fluorescence plate reader using the appropriate filters.

 

Precautions:

1. Since Calcein-AM is very sensitive to humidity, the solution must be tightly sealed after each use. It is recommended to aliquot and seal for storage according to the amount used in a single experiment.

2. Due to the poor stability of Calcein-AM, this staining working solution must be prepared fresh and used on the same day.

3. Propidium Iodide (PI) is carcinogenic to some extent, so protective measures must be taken during handling. If it comes into contact with the skin, it should be immediately washed off with tap water.

4. For your safety and health, please wear a lab coat and disposable gloves during the operation.

 

Staining Result Display:

 

I. Bright-field image of human hepatocellular carcinoma organoids

The amplification process of human hepatocellular carcinoma organoids (from few to many).

 

II. Video of live-dead staining of human hepatocellular carcinoma organoids.

 

III. Images of live-dead staining of human hepatocellular carcinoma organoids.

Human hepatocellular carcinoma organoid live cell, dead cell, and merged live-dead cell staining images

Human hepatocellular carcinoma organoid live-dead cell staining merged image

 

Item NO.	Product Name	Size
abs50056	Calcein-AM/PI Double Stain Kit	500T
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

 

Absin provides antibodies, proteins, ELISA kits, cell culture, detection kits, and other research reagents. If you have any product needs, please contact us.

Absin Bioscience Inc. Email: worldwide@absin.cn

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