Clinical Chemistiy 3 19-325
42:2
Immunology
(1996)
Use of flameless atomic absorption spectroscopy in immune cytolysis for nonradioactive determination of killer cell activity PAOLA
BoRELI,l,2*
ANNALISA
BARGELLINI,1
STEFANO
spectroscopy (GF-AAS). This technique may be adopted for use in laboratories equipped with electrothermal atomic absorption spectrometers. Nonradioactive Cr as Na2CrO4 was used to label target cells (1(562), and cell lysis was evaluated by measuring Cr released after 4 h of incubation with the effectors. We selected 520 gxgfL as the optimal dose for labeling targets, between 12 and 20 h as the optimal incubation time, and i0 cells as the optimal target size. Advantages of this method include: (a) exclusion of radioactive tracer, with no risk for workers; (b) limited costs; (c) high sensitivity and reproducibility; (d) possibility to store samples; and (e) better control of Cr used for labeling cells due to well-determined, fixed Cr concentrations in the range of nontoxic and linear cellular uptake. Comparison with data obtained by conventional 51Cr labeling of targets killed by the same effectors was excellent, yielding comparable results and corroborating the method. m1ti’rs:
cytotoxicity
#{149} chromium
uptake
.
and
ANDREA
COSSARIZZA2
environmental and basic sciences, epidemiology, preventive medicine, and toxicology [16-26]. We have developed a safe, nonradioactive procedure for the detection of N’K cell cytolysis by measuring cell-released metal by graphite furnace atomic absorption spectroscopy (GF-AAS). This technique could be routinely adopted in any type of laboratory, and research laboratories, even if authorized to use radioactive reagents, could move away from radioactive techniques, especially if they are already equipped with GF-AAS. To our knowledge, the measurement of MCr released from target cells is the only widely adopted method for evaluating NK cell activity [2 7-29]. Potential alternatives such as the use of fluorescent markers [30-32], evaluation of endogenous enzymes
We describe here a novel method to evaluate natural killer (NK) cytolytic activity by use of flameless atomic absorption
INDEXING
SALvIou,2
released from the cells /33, 34], and automated colorimetric assays [35] are still in experimental stages. Although other radioactive tracers, e.g., 75Se, have been used [36, 37], the only nonradioactive element proposed for this use is Eu, which appears promising for high sensitivity and rapidity [38-40]. At present, however, its cost is prohibitive. We propose here a method based on labeling target cells (K562) with nonradioactive Cr as Na,Cr04 and evaluating the release of the label by cell lysis in terms of absolute metal concentration. Compared with the conventional 1Cr assay, our method has several advantages, particularly avoidance of the radioactive tracer, which emits gamma rays and presents health hazards for workers. Furthermore, Na,Cr04 is inexpensive, does not require special facilities and waste disposal, and has unlimited storage time. In a previous paper, we validated the proposed method by comparing results of the “cold” assay with the radioactive Cr assay for each individual [41]. Here, we describe in detail how the released Cr can be accurately measured by GF-AAS. We also compare the calibration curve for NK cytotoxicity obtained with the new method with an independent calibration curve obtained by using 5tmCr. The very high sensitivity of this technique has been previously demonstrated in evaluations of uptake and accumulation of various metals, including Cr, by human lymphocytes under several experimental conditions [4244]. The optimal labeling conditions and the rate of Cr uptake by targets are analyzed and described in detail.
immune
system The evaluation of natural killer (NK) cell activity has been included in a battery of assays used in epidemiological studies to detect early derangements in the immune system associated with several diseases [J_4]3 Indeed, acute and chronic stress [5-7], intense physical activity [8-10], exposure to toxic substances [11], and unhealthy life-styles [12-15] are associated with modifications of immunological cytotoxic responses. These studies have received a growing interest, prilnarily among researchers in
Sections of Hygiene and Microbiology and 2 General Pathology, l)epartment of Biomedical Sciences, University of Modena, Via Campi 287, 41100 Modena, Italy. “Author for correspondence. Fax +39 59 363057; e-mail
[email protected]. Nonstandard abbreviations: NK, natural killer; GF-AAS, graphite furnace atomic absorption spectroscopy; PBS, phosphate-buffered saline; HBSS, flanks balanced salt solution; and E:T, effector:target cell ratio, Received August Il, 1995; accepted November 9, 1995.
319
320
Borella
Materials
et al.: GFAAS/Cr
determination
and Methods
MATERIALS
All glassware, instruments, and reagents used both in target cell maintenance and during execution of the NK assay were considered possible sources of contamination and were therefore accurately monitored for their Cr content. We avoided using glassware, glass pipettes, and items composed of metal. No detectable amounts of Cr were leached after 24-h contact with water by tissue culture flasks (Greiner, Frickenhausen, Germany), polypropylene sterile test tubes with colorless polypropylene stoppers (Multilab, Zoersel, Belgium), 96-well roundbottom microplates (Corning Glass Works, Corning, NY), disposable pipette tips (Eppendorf, Hamburg, Germany), and polystyrene
sample
cups.
REAGENTS
Water used to prepare calibrators, phosphate-buffered saline [PBS (pH 7.4): 10 mmol/L phosphate and physiological saline (150 mmol/L NaCI)], and saline were purified by using a MilliQ (Millipore, Medford, MA) water purifier on predeionized water. Hanks balanced salt solution without Ca2 and Mg2 (HBSS), RPMI 1640, fetal calf serum, and antibiotics were purchased from Gibco BRL (Paisley, U’K); Na,Cr04, Nonidet P40, HCI, and HNO5 were from BDH (Poole, UK). Concentrations of Cr in complete medium were 1.11 ± 0.22 .tg/L (0.02 tmol/L) and in reagents were: 0.8 ± 0.15 jg/L (0.015 tmol/L) in PBS, 1.1 ± 0.19 tg/L (0.02 mol/L) in HBSS, 0.2 ± 0.04 g/L (0.004 j.mol/L) in physiological saline, and
