Calcein-Acetyoxymethyl Cytotoxicity Assay: Standardization of a Method Allowing Additional Analyses on Recovered Effector Cells and Supernatants

Abstract

Cytotoxicity assays provide an in vitro evaluation of the lytic activity of NK and T cells against tumors or transformed cells. However, none of these methods allow the recovery of cells or supernatants after the assay. We standardized a microcytotoxicity test using calcein-acetoxymethyl (calcein-AM) dye that requires very small quantities of cells while maintaining the same sensitivity as the traditional 51 Cr assay. The assay is applicable to resting as well as activated human effector cells and uses different targets such as human cell lines that are adherent or growing in suspension and resistant or sensitive. The most important feature of the method is the possibility of recovering cells and supernatants for additional analyses such as phenotyping and evaluation of soluble factors.Cytotoxicity assays provide an in vitro evaluation of the lytic activity of natural killer (NK) and T cells against tumors or transformed target cells (3,16,25). In in vitro experimental conditions, lytic activity is evaluated using isotopes or dyes either released from dead cells or retained by living ones. The 51 chromium ( 51 Cr) release assay is the most widely used method although it has several significant drawbacks. The major disadvantage results from the use of a radioactive compound, with the related problems of handling and disposal due to the short half-life of the isotope.To overcome these problems, several nonradioactive methods have been developed, but none have yet found a broad acceptance, probably due to a lack of comparability of their results with the results obtained using the 51 Cr release assay, which still remains the most popular. Some alternative methods consist of measuring endogenous or transfected reporter enzymes released in the supernatant by dead targets (1,11,19,24). Other assays using nonradioactive compounds (dimethylthiazol-diphenyl bromide tetrazolium bromide, methylumbelliferyl heptanoate, Alamar blue) evaluate the variations in metabolic activity, which is directly proportional to the number of viable cells (6, 18). Alternative methods using fluorochromes (e.g., europium, D275, rhodamine-123, carboxyfluorescein diacetate, bis-carboxyethyl-carboxyfluorescein) (7,9,12,14,23,26,28) measure the amount of dye released from or remaining in prelabeled target cells (2, 8, 13). The major drawbacks for these methods appear to be the high spontaneous release of the fluorescent dye, the slow specific release of the fluorescein dye, and the low intensity of the fluorescence signal, all of which decrease the overall sensitivity of the test.Among fluorescent dyes, the use of calcein-acetoxymethyl (calcein-AM) in cytotoxicity assays has already been described (13,21,22). This dye has good retention in targets and low pH sensitivity, and there is no stain transfer among cells (21). Acetoxymethyl ester of calcein is a lipid-soluble diester fluorogenic esterase substrate that passively crosses the cell membrane and that is frequently used to stain viable cells. Inside the cells it is converted by intracellular ...

Mitochondrial Alterations and Oxidative Stress in an Acute Transient Mouse Model of Muscle Degeneration

“…The dependence of Rhod-2 fluorescence on Ca 2ϩ influx was determined by incubating the cells with CTX Ϯ 2 mM EGTA, followed by quantitation of fluorescence in a fluorimeter (Tecan). Determination of Membrane Damage-Cells grown in 60-mm dishes to 70% confluence were loaded with 5 M calcein AM for 45 min at 37°C in HEPES buffer (20). The cells were washed three times with HEPES buffer and incubated with CTX (2 M) for 0 -30 min.…”

Loss of Host Type-I IFN Signaling Accelerates Metastasis and Impairs NK-cell Antitumor Function in Multiple Models of Breast Cancer

“…Standard 4-hour cytotoxicity assays were completed as described elsewhere (22). Briefly, splenic NK cells from na€ ve or poly(I:C) activated (250 mg i.p.…”

Individual Vγ2-Jγ1.2+ T cells respond to both isopentenyl pyrophosphate and Daudi cell stimulation: generating tumor effectors with low molecular weight phosphoantigens

“…To quantify the cytotoxic capacity of Vγ2Vδ2 T cells we used a non-radioactive, fluorometric cytotoxicity assay involving the dye calcein-acetoxymethyl (calcein-AM) [32]. Tumor targets were labeled for 15 minutes with 2μM calcein-AM (Molecular Probes, Eugene, Oregon) at 37°C in 5% CO 2 and then washed once with phosphate-buffered saline (PBS).…”