Calcein-AM (Calcein acetoxymethyl ester)

Calcein-AM is a highly lipophilic vital dye that rapidly enters viable cells. It is converted by intracellular esterases to calcein that produces an intense green (530-nm) signal, and is retained by cells with intact plasma membrane.^[1]

In vitro experiment it shown that calcein-AM assay used to assess human RBC viability after incubation (37°C for 3 and 20 h) in the presence of Ca2+ (2.5 mM) and ionophore A 23187 (0.5 μM).^[1] 0.05 μM was the optimal concentration of CAM (Calcein-AM) for staining effector cells by testing 0.05, 0.1, 0.2, and 0.4 μM. Using 0.05 μM CAM to stain the PBMCs and expanded NK cells from three normal volunteers, the results demonstrated that there is no significant decrease in cytotoxicity and CAM staining had no significant effect on human NK cell activity in PBMCs or in expanded NK cells.^[2] In vitro, 50μm calcein AM's fluorescent signal of 1 x lo5 lymphocytes was close to the saturation level, while the signal emitted by lymphocytes labeled with 20μm calcein AM was only slightly lower. ^[3] Calcein-AM has cytotoxic activity against human tumor cell lines (such as the human lymphoma U-937-GTB) at low concentrations (2.5 ug/ml).^[4] In vitro experiment it demonstrated that in the mixed macrophages and THP-1 cells (5x105 cells/ml), Calcein-AM (2 µM)/propidium Iodide (PI) (4.5 µM) staining assay Calcein-AM/PI double staining was used to quantify the number of living and dead cells as a cell death assay.^[5] In addition, The cells in OA chondrocytes were seeded in 24-well plates (2 × 104 cells/well), cultured for 4 h, the cells were treated with 5 μL Calcein-AM (2 μM) and 5 μL PI (2 μM) at 37°C in conditions void of light for 30 min, and then analyzed under a fluorescence microscope.^[6]

References:
[1].Bratosin D, et al. Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging. Cytometry A. 2005 Jul;66(1):78-84.
[2].Jang YY, et al. An improved flow cytometry-based natural killer cytotoxicity assay involving calcein AM staining of effector cells. Ann Clin Lab Sci. 2012 Winter;42(1):42-9.
[3].Braut-Boucher F, et al. A non-isotopic, highly sensitive, fluorimetric, cell-cell adhesion microplate assay using calcein AM-labeled lymphocytes. J Immunol Methods. 1995 Jan 13;178(1):41-51.
[4].Liminga G, et al. Cytotoxic effect of calcein acetoxymethyl ester on human tumor cell lines: drug delivery by intracellular trapping. Anticancer Drugs. 1995 Aug;6(4):578-85.
[5].Xiang N, et al. Gardnerella vaginalis induces NLRP3 inflammasome-mediated pyroptosis in macrophages and THP-1 monocytes. Exp Ther Med. 2021 Oct;22(4):1174.
[6].Zhang L, et al. MicroRNA-140-5p represses chondrocyte pyroptosis and relieves cartilage injury in osteoarthritis by inhibiting cathepsin B/Nod-like receptor protein 3. Bioengineered. 2021 Dec;12(2):9949-9964.

Calcein-AM是一种高脂溶性的活细胞染料,能够快速进入存活的细胞。它被细胞内酯酶转化为卡尔西因,产生强烈的绿色（530纳米）信号,并被具有完整质膜的细胞保留。

实验室内的实验证明,使用荧光染料Calcein-AM可以评估人类红细胞在存在Ca2+（2.5 mM）和离子载体A 23187（0.5 μM）下孵育后（37°C,3小时和20小时）的存活率。测试了0.05、0.1、0.2和0.4μM浓度的CAM,结果显示0.05μM是染色效果最佳的浓度。使用0.05μM CAM对三名正常志愿者PBMCs和扩增NK细胞进行染色,结果表明,在PBMCs或扩增NK细胞中,Cytotoxicity没有显著降低,并且CAM染色对人类NK细胞活性没有显著影响。在体外实验中,50μm Calcein AM荧光信号接近饱和水平时1 x lo5淋巴细胞标记物发出的信号略微较低。Calcein-AM对人类肿瘤细胞系（如人淋巴瘤U-937-GTB）具有毒性作用,在低浓度下（2.5ug/ml）。在混合巨噬细胞和THP-1 细胞 (5x105 cells/ml) 的体外实验中, 使用 Calcein-AM(2 µM)/propidium Iodide (PI) (4.5 µM) 染色法进行 Calcein-AM/PI 双重染色,以定量活细胞和死亡细胞的数量作为细胞死亡检测方法。此外,在OA软骨细胞中种植2×104个细胞/well,培养4小时后,在无光条件下将5μL Calcein-AM（2μM）和5μL PI（2μM）处理在37°C下30分钟,并在荧光显微镜下分析。