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Cat. No.: 3DCAM-1 (for 0.1ml)

Cat. No.: 3DCAM-5 (for 0.5ml)

Cat. No.: 3DCAM-20 (for 2ml)

Description

SBS3D™ Calcein AM Staining Solution (100X) is a quick and convenient solution for staining live cells in 3D cultured cell spheroids or organoids. With just 10 minutes of staining, bright green fluorescence can be observed in live cells under a fluorescence microscope.

Calcein AM (Calcein Acetoxymethyl Ester), also known as calcein AM or calcein acetoxymethyl ester in Chinese, is a green fluorescent probe that can permeate cells and is commonly used to measure eukaryotic cell viability or mitochondrial permeability transition pore (MPTP). In recent years, it has also been widely used in cell viability, cytotoxicity, and cell proliferation assays.

Calcein AM is derived from calcein by adding an acetoxymethyl (AM) ester group, enhancing its hydrophobicity. This allows it to easily penetrate the membranes of live cells and enter the cells, thus labeling them. Calcein AM itself is non-fluorescent. Once inside the cell, endogenous esterases hydrolyze it into calcein, a polar molecule with a strong negative charge that cannot permeate the cell membrane, thus being retained inside the cell. Calcein emits a strong green fluorescence. Compared to other similar probes, calcein AM has very low cytotoxicity, minimally affecting cellular functions such as cell proliferation or lymphocyte chemotaxis, and has low pH sensitivity. This makes calcein AM one of the best fluorescent probes for staining live cells.

Since dead cells lack esterases, calcein AM is used only for viability testing and short-term labeling of live cells. Propidium iodide (PI), a red fluorescent nucleic acid dye that cannot penetrate the membranes of live cells and stains only dead cells with compromised membrane integrity, is often used in combination with calcein AM for dual fluorescence staining of live and dead cells.

Calcein AM can be applied to most mammalian cells. Reports have shown that calcein AM can also be used in certain plant cells, such as root border-like cells of Arabidopsis, and certain yeasts, such as Pichia anomala and Saccharomyces cerevisiae. Due to the composition of their cell membranes, calcein AM cannot enter live cells of some parasites, such as Leishmania, but can enter early apoptotic parasite cells, allowing it to be used with PI to detect early apoptotic parasites. Calcein AM is not suitable for fungi and bacteria due to their cell walls, which prevent calcein from entering the cells.

Calcein itself is a metal chelating indicator, and its fluorescence signal is quenched when complexed with metal ions such as Co2+, Ni2+, Cu2+, Fe3+, and Mn2+ under physiological pH conditions.

Calcein AM has a molecular formula of C46H46N2O23, a molecular weight of 994.86, and a CAS number of 148504-34-1. The hydrolyzed product calcein has a maximum excitation wavelength of 494nm and a maximum emission wavelength of 514nm.

Based on the requirement of 1μl of SBS3D™ Calcein AM Staining Solution (100X) per well in a 96-well plate, this product's 0.1ml packaging can test 100 samples.

Features

• This product has a wide range of applications. It can be used for 3D cell spheroids or organoids cultured by conventional methods, including ultra-low attachment plates, Matrix-Gel or Matrigel-coated plates, agarose-coated plates, hanging drop culture plates, etc.
• The product is easy to use, with the entire detection process taking approximately 10-30 minutes. After treating 3D cell spheroids with apoptosis inducers or other treatments, simply add this product and incubate in the dark for 10 minutes to proceed with fluorescence detection.

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Storage

Store at -20ºC protected from light for one year.

Precautions

• Repeated freeze-thaw cycles may reduce staining efficacy. To ensure optimal use, avoid repeated freeze-thaw cycles and consider aliquoting after the first thaw for storage.
• Cell spheroids may deform or disperse under external forces. Perform PBS washing and liquid changes gently to avoid damaging or dispersing 3D cell spheroids.
• Different types of cell spheroids may have varying tolerances to apoptosis inducers. After inducing apoptosis in 3D cell spheroids, their morphology may change. Observe the cell spheroid morphology under a microscope before staining and consider selecting more intact cell spheroids for staining analysis.
• The concentration of this product has been optimized by SBS3D to meet the needs of live cell staining and conventional staining. If a specific concentration of Calcein AM is required, please purchase Calcein AM.
• Serum and phenol red in the culture medium can affect the staining of SBS3D™ Calcein AM Staining Solution (100X). It is recommended to wash the cell spheroids once before adding SBS3D™ Calcein AM Staining Solution (1X).
• Cells labeled with Calcein AM exhibit uniform fluorescence and are effective for short-term cell migration tracking. However, the duration of fluorescence varies with cell type, culture conditions, and other factors, generally lasting within a few hours, and may be rapidly expelled by certain cell types. For long-term cell labeling, use fluorescent probes such as CFDA SE (C1031).
• Fluorescent dyes are prone to quenching, so it is recommended to complete the detection on the same day of staining.
• This product is intended for scientific research use by professionals only and is not for clinical diagnosis or treatment, food, or drugs. Do not store in residential areas.
• For your safety and health, wear a lab coat and disposable gloves while handling.