Measurement of CTL-induced cytotoxicity: The caspase 3 assay

Cytotoxic T lymphocytes (CTL) are critical effector cells of the immune system. Measurement of target cell damage has historically been an important measure of CTL function. CTL kill their target cells predominantly by inducing programmed cell death, or apoptosis. The gold standard for CTL-mediated cytotoxicity has been the ⁵¹Cr release assay. However, measurement of target cell lysis by ⁵¹Cr release does not provide mechanistic information on the fate of target cells, especially at the single cell level. Given the recent advances in our understanding of programmed cell death, newer assays are required which evaluate the status of the apoptotic pathways in target cells. We have developed a flow cytometry-based assay for CTL-mediated cytotoxicity based on specific binding of antibody to activated caspase 3 in target cells. Our assay is convenient and more sensitive than the ⁵¹Cr release assay. The use of this assay should allow mechanistic studies of the intracellular events resulting from CTL attack.     

The combined effect of lymphokine activated killer cell and radiation therapy on rat brain tumorin vitro

Thein vitro effect of a combined treatment with lymphokine activated killer (LAK) cell and radiation therapy on rat brain tumor was examined using⁵¹Cr release assay. The tumor cell-line used in this experiment was 9L rat brain tumor derived from a Fischer 344 rat. LAK cells were obtained by culturing rat lymphocytes with recombinant human interleukin 2 for at least 3 days. The cytotoxic activity of the LAK cells was examined by⁵¹Cr release assay. Irradiation was done by exposing the microtiter plate in which the¹⁵Cr labeled 9L cells and LAK cells were cultured to a¹³⁷Cs gamma cell unit. Without irradiation, there was 18% cytotoxicity in the 1:100 tumor-to-LAK cell ratio specimen after 24 hrs cocultivation. However, if 5 Gy of irradiation was given, followed by 12 hrs incubation, the cytotoxicity was enhanced significantly at the same cell ratio (30%). This enhancement effect was the most prominent when the cell ratio was 1:100 and the irradiation dose was 5 Gy. To generate the enhancement effect, an incubation time of over 8 hrs both before and after irradiation was required. The supernatant of the LAK cells showed 19.8% and 11.4% cytotoxicity with and without irradiation, respectively. This result indicates the participation of a cytotoxic factor released from LAK cells.This work is supported in part by grant from Univeristy of Tsukuba Project Research.     

Differential 51Cr uptake of human peripheral lymphocytes separated by density gradient electrophoresis

Human peripheral lymphocytes were labeled with ⁵¹Cr before or after separation by preparative density gradient electrophoresis. In both cases, wide variations in the distribution of ⁵¹Cr in the electrophoresed cells was observed. In general, there was significantly more ⁵¹Cr per cell in the high mobility fractions. These results suggest caution in the interpretation of cytotoxic assays where ⁵¹Cr-labeled lymphocytes are used as target cells and prompt further studies by different separation methods.     

Uptake and retention in suckling rats of51chromium fed with human milk or infant formulas

Optimum concentration of Cr for infant formulas has not been established. Such components as soy protein or supplemental Fe could influence absorption and retention. Suckling rat pups were used to evaluate the influence of three commercial formulas and human milk, all of which had been incubated with⁵¹CrCl₃ for 1 h, on the uptake and retention of the added⁵¹Cr. After fasting 3 h, the pups were intubated with a single dose of 25 μCi⁵¹CrCl₃ in either a cow's milk-based formula, an Fe-supplemented cow's milk-based formula, a soy-based formula, or human milk. Six hours later,⁵¹Cr was counted in five organs, thymus, blood, and total urine. Absorption of⁵¹Cr was low. At 6 h, percent⁵¹Cr in blood was <0.2% of the dose, and total⁵¹Cr excretion in urine was <1.8%. The uptake and retention of⁵¹Cr and its concentration in any of the organs, thymus, blood, and urine were not influenced by different types of formula or by human milk.     

Adjuvant effect of poly(A:U) upon T cell-mediated in vitro cytotoxic allograft responses

The effect of complexes of polyadenylic acid and polyuridylic acid [poly(A:U)] on thymus-processed lymphocytes was studied using a tissue culture system in which T cells responded to cell bound alloantigens. The in vitro activation of T cells into cytotoxic lymphocytes was assessed with the aid of the ⁵¹Cr cytotoxic assay. Introduction of poly(A:U) into cultures or pretreatment of thymus cells prior to culture resulted in a reduction in the time required for the development of maximal cytotoxic activity as well as a reduction in the dose of allogeneic cells required for maximum stimulus. Poly(A:U) had no influence on the ability of differentiated cytotoxic T cells to lyse ⁵¹Cr-labeled target cells. The amplification of cytotoxic activity caused by poly(A:U) was specific to the antigens used to activate the thymus lymphocytes.     

Spontaneous51Cr release by isolated rat hepatocytes: An indicator of membrane damage

Summary Radiochromium uptake and release by isolated rat hepatocytes in suspension was monitored under continuous-labeling conditions. Cell protein remained unchanged during the absorption phase, whereas the release of⁵¹Cr correlated well with the loss of cell viability and release of cytoplasmic protein. The results suggest that under equilibrium conditions,⁵¹Cr release represents an efflux of label from damaged or dying preparations and not an elution of radioisotope from intact cells.     

A novel 96-well scintillation proximity assay for the measurement of apoptosis

The translocation of phospholipids across the plasma membrane has been widely documented as one of the earliest measurable biochemical events of apoptosis. Using fluorescently labelled annexin V, which preferentially binds phosphatidylserine (PS) in the presence of Ca²⁺, the externalization of PS can be measured and apoptosis quantified using flow cytometry. Conventional detection methods utilizing annexin V, while faster than in situ DNA end-labelling or DNA laddering, require extensive sample preparation which may compromise samples and makes rapid, high volume screening prohibitive. This paper describes a novel assay for the measurement of apoptosis based upon binding of radiolabelled annexin V to apoptotic cells attached to the growth surface of a 96-well scintillating microplate (Cytostar-T®). We compared measurements of apoptosis made by flow cytometry to those obtained with the scintillating microplate in three model systems, treatment of: mouse connective tissue (L-M) cells with lymphotoxin (LT), human lung carcinoma (H460) cells with Apo-2 ligand and human umbilical vein endothelial (HUVE) cells with staurosporine. In this assay, we compare both direct and indirect labelling methods by utilizing either iodinated annexin V or biotinylated annexin V/[³⁵S] streptavidin to radiolabel apoptotic cells. The signal detected is a direct consequence of the binding of annexin V to externalized PS on apoptotic cells and the proximity of the label to the base of the plate. Using this method, separation of bound and unbound radiolabel signal occurs directly within the well resulting in a sensitive assay that requires minimal manipulation and can accomodate a large number of samples.     

The influence upon mitogenic and cellular immunologic reactive systems in vitro by poly(I:C) and BCG murine interferons induced in vivo.

We have previously reported that lymphocytes from W/Fu rats immunized with syngeneic (C58NT)D tumor cells were cytotoxic against these cells in a 4-hr ⁵¹Cr release assay. We have investigated the feasibility of cryopreserving lymphocytes and target cells and have selected freezing conditions which provide good yields of viable cells and functional activity. Lymphocytes from different animals had a recovery of 60–80% viability which resulted in a corresponding 55–75% recovery of cytotoxic activity. Repeated testing of lymphocyte cytotoxicity from a pool of frozen spleen cells against either fresh or frozen (C58NT)D cells gave reproducible cytotoxicity. In addition, recovery of high levels of lymphocyte function was also demonstrated when cryopreserved cells were employed in long-term cytotoxic assays, i.e., ³H-proline and ¹²⁵IUdR release assays, in the lymphoproliferative response to mitogens (PHA and Con A)³ or tumor cells (MLTI) as measured by ³H-thymidine incorporation, and in the in vitro generation of secondary cytotoxicity.By employing these cryoprotective techniques it is possible to have: 1) a population of lymphoid cells with known functional activity and 2) a pool of target cells with known susceptibility to lysis and antigenic content. Furthermore, the use of frozen cells as internal standards in each test also permits the analysis of assay variation as well as the study of variation in various cell types.     

Cytological and functional analysis of inflammatory infiltrates in human malignant tumors: III. Further functional investigations using cultured autochthonous tumor cell lines and freeze-thawed infiltrating inflammatory cells

Short-term monolayer cultures, dominated by cells with malignant characteristics, were established from human tumors displaying an unusually strong host-inflammatory response. Upon repeated testings in the ⁵¹Cr release cytotoxicity assay, blood leukocytes were frequently cytotoxic (a) to autologous and allogeneic tumor cells, without any apparent restriction as to tumor origin or HLA type, and (b) to the so-called natural killer (NK) target cells. The anti-tumor cytotoxicity disappeared with time. The in situ inflammatory cells, freshly isolated or recovered from the deep freeze, did not display any type of cytotoxic activity. Nor were they notably suppressive to either type of blood leukocyte cytotoxic activity in the ⁵¹Cr release assay. Cytological analysis demonstrated that the “large granular lymphocytes” (LGL), known to be largely responsible for the NK activity in man, were prominent in the blood but not in the inflammatory infiltrate. These preliminary observations suggest that lack of cytotoxic activity in situ correlates with the absence of effector cells in the inflammatory infiltrate.     

Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the radionuclide Chromium 51 (⁵¹Cr) to label target cells. These targets are then exposed to effector cells and the release of ⁵¹Cr from target cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of ⁵¹Cr. As effector cells, we used an activated autoreactive clonal population of CD8⁺ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with ⁵¹Cr labeled target NIT cells for 16 hours, release of ⁵¹Cr was recorded to calculate specific lysis Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37°C, with 5% CO₂, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.     

A fluorometric assay of SIRT1 deacetylation activity through quantification of nicotinamide adenine dinucleotide

Sirtuins are nicotinamide adenine dinucleotide (NAD⁺)-dependent deacetylases that catalyze the deacetylation of proteins such as histones and p53. A sensitive and convenient fluorometric assay for evaluating the SIRT1 enzymatic activity was developed here. Specifically, the remaining NAD⁺ after the deacetylation was determined by converting NAD⁺ to a highly fluorescent cyclized α-adduct compound. By this assay, we found that nicotinamide, Cu²⁺, and Zn²⁺ antagonize the activity of SIRT1. Resveratrol stimulates the enzymatic activity specifically with 7-amino-4-methylcoumarin (AMC)-labeled acetylated peptide. Epigallocatechin galate (EGCG) inhibits SIRT1 activity with both AMC-labeled and unlabeled peptide. However, a combination of vitamin C with EGCG can reverse the inhibition of EGCG with the unlabeled peptide or stimulate the deacetylation of AMC-labeled peptide by SIRT1. The assay does not require any isotopic material and thus is biologically safe. It can be adapted to a 96-well microplate for high-throughput screening. Notably, the acetylated peptides with or without fluorescent labels may be used in the assay, which facilitates the substrate specificity study of SIRT1 activators or inhibitors in vitro.     

Protein-free culture medium improvement: testing additives and their interactive effects in 96-well plates

In order to achieve a rational screening of additives suspected to improve antibody production over basal Dulbecco's Modified Eagle Medium (DMEM), we have developed a 96-well plate method for simultaneously testing individual and interactive effects of multiple additives. Cell viabilities were determined directly in each well by a colorimetric assay (MTT assay) and antibody production was measured by an enzyme-linked immunosorbent assay (ELISA) performed on well supernatants. Such as supplemented culture medium might considerably reduce production costs since commercial serum- or protein-free media are sold at serum-enriched basal medium costs. The CBM-P22 mouse cell line used in this study was shown to be sensitive to key amino acids, oxalacetic acid, ethanolamine and selenium at low cell density (<1 × 10⁵ cells·ml^–1). When these cells were inoculated in 96-well plates their antibody productivity was improved (sevenfold) by adding these additives to the basal DMEM as evidenced by ELISA absorbance readings. This improved productivity, obtained with the supplemented DMEM, named DOWSENs, is comparable to the one obtained with the commercial, but costly, protein-free hybridoma medium (PFHM II), taken here as the positive control. Results were confirmed by growing these cells in different media in standard (25 cm²) T-flask static culture. In addition, specific amino acid consumption, as analysed by HPLC, showed that asparagine and tryptophan when added to DMEM may improve antibody production, even if these amino acids are not limiting or highly consumed in PFHM II. Using this 96-well plate assay allows the assessment of a large number of different assays with a few milligrams of the product(s) tested. This is a rapid technique that gives results for additives that are not easily quantified by analytical techniques or for the study of interactive effects, which is time consuming and labour-intensive when done in individual T-flasks. Correspondence to: J. Côté     

New Instruments for High Throughput Receptor Binding Assays

Abstract

The TopCount^(R) Microplate Scintillation Counter and the Matrix 9600^(R) Direct Beta Counter are microplate compatible instruments developed to meet the needs of investigators using radioisotope assays adapted for very high throughput. This paper describes these instruments and their application to receptor binding assays. When combined with the appropriate sample handling equipment and filter media, use of these multi-detector instruments improves sample handling efficiency and shortens overall counting time. The assay protocols including filtration through glass fiber mats and membrane filters have been investigated. Results obtained from these new instruments are compared to standard techniques using conventional liquid scintillation and gamma counting.     

Announcements

Human peripheral blood monocytes were reproducibly shown to lyse a variety of tumor cells in a 3- to 4-hr ⁵¹Cr release assay. Ficoll-Hypaque-purified mononuclear cells were suspended in medium supplemented with either 10% autologous serum or fetal calf serum (PCS). With either serum, highly purified (97–99%) and viable (>99%) monocyte suspensions were obtained by EDTA-reversible adherence to plastic surfaces which had been precoated with autologous serum. When used as effectors in cytotoxicity assays, the monocytes recovered from mononuclear cells suspended in FCS-supplemented medium exhibited higher cytolytic activity and were therefore used for further studies. Using FCS for both coating the plates and supplementing the suspension medium resulted in monocytes with low cytolytic activity. Tumor cell lysis measured by ⁵¹Cr release was detected within 2 hr of incubation and increased gradually with time. The level of lysis was dependent on the effector/target ratio and the tumor target cell employed. The involvement of natural killer lymphocytes in the observed tumoricidal activity was excluded. Detection of cytotoxic activity in a short-term assay will be very helpful in further studies of the mechanism of tumor cell killing by human monocytes since potential complicating effects of long-term in vitro cultivation will be minimized.     

Continuous,label-free, 96-well-based determination of cell migration using confluence measurement

Cellular migration is essential in diverse physiological and pathophysiological processes. Here, we present a protocol for quantitative analysis of migration using confluence detection allowing continuous, non-endpoint measurement with minimal hands-on time under cell incubator conditions. Applicability was tested using substances which enhance (EGF) or inhibit (cytochalasin D, ouabain) migration. Using a gap-closure assay we demonstrate that automated confluence detection monitors cellular migration in the 96-well microplate format. Quantification by % confluence, % cell free-area or % confluence in cell-free area against time, allows detailed analysis of cellular migration. The study describes a practicable approach for continuous, non-endpoint measurement of migration in 96-well microplates and for detailed data analysis, which allows for medium/high-throughput analysis of cellular migration in vitro.     

A novel method for producing target cells and assessing cytotoxic T lymphocyte activity in outbred hosts

Background  

Cytotoxic T lymphocytes play a crucial role in the immunological control of microbial infections and in the design of vaccines and immunotherapies. Measurement of cytotoxic T lymphocyte activity requires that the test antigen is presented by target cells having the same or compatible class I major hystocompatibility complex antigens as the effector cells. Conventional assays use target cells labeled with ⁵¹chromium and infer cytotoxic T lymphocyte activity by measuring the isotope released by the target cells lysed following incubation with antigen-specific cytotoxic T lymphocytes. This assay is sensitive but needs manipulation and disposal of hazardous radioactive reagents and provides a bulk estimate of the reporter released, which may be influenced by spontaneous release of the label and other poorly controllable variables. Here we describe a novel method for producing target in outbred hosts and assessing cytotoxic T lymphocyte activity by flow cytometry.     

Use of the enriched stable isotope Cr-50 as a tracer to study the metabolism of chromium (III) in normal and diabetic rats

The activable enriched stable isotope Cr-50 compound Cr₂O₃ was used as a tracer to study the metabolism of chromium(III) [CR(III)] intragastrically administered in normal and diabetic rats. The comparison of absorption, distribution, and excretion in organs and tissues of the two groups do not show much alteration, but some differences exist indeed. The contents of⁵¹Cr radioactivity of the diabetic rats appear to be of higher retention than in most studied organisms. The urinary⁵¹Cr excretion of diabetics is significantly higher than that of normal rats. Therefore, a conclusion can be drawn that the insulin-dependent rats generally absorb and excrete more chromium (Cr) than the normal rats.     

Optimization of a phagocyte microplate chemiluminescent assay

Chemiluminescent assays have been used to quantify phagocytic activity since 1972. In recent years these assays have been adapted to the 96-well microplate format as new luminometers have been developed. In this report we describe the optimization of a lucugenin enhanced phagocyte chemiluminescent assay using a Titertek Luminoskan. Factors such as cell concentration, serum concentration in the opsonization of the zymosan used and lucigenin concentration were all optimized in our assay. In addition we have found that some of the unique features of the Luminoskan, continuous microplate agitation during the assay and microplate temperature control up to 43°C, also significantly enhanced the chemiluminescent response.     

Increased efficiency for performing colony formation assays in 96-well plates: novel applications to combination therapies and high-throughput screening

The colony formation assay (CFA) is the gold standard for measuring the effects of cytotoxic agents on cancer cells in vitro; however, in its traditional 6-well format, it is a time-consuming assay, particularly when evaluating combination therapies. In the interest of increased efficiency, the 6-well CFA was converted to a 96-well format using an automated colony counting algorithm. The 96-well CFA was validated using ionizing radiation therapy on the FaDu (human hypopharyngeal squamous cell) and A549 (human lung) cancer cell lines. Its ability to evaluate combination therapies was investigated by the generation of dose-response curves for the combination of cisplatin and radiation therapy on FaDu and A549 cells. The 96-well CFA was then transferred to a robotic platform for evaluating its potential as a high-throughput screening (HTS) readout. The LOPAC1280 library was screened against FaDu cells, and eight putative hits were identified. Using the 96-well CFA to validate the eight putative chemicals, six of the eight were confirmed, resulting in a positive hit rate of 75%. These data indicate that the 96-well CFA can be adopted as an efficient alternative assay to the 6-well CFA in evaluating single and combination therapies in vitro, providing a possible readout that could be used on a HTS platform.     

Lymphocyte-mediated cytotoxicity to autologous cells infected with measles virus. II. Specificity of the cytotoxic reaction and characterization of the effector cells involved.

The mechanism of lymphocyte-mediated cytotoxicity to cells infected with measles virus was investigated. Cytotoxicity was measured in a direct assay, immediately after the isolation of lymphocytes from human peripheral blood; mononuclear leukocytes, infected with measles virus in vitro, served as autologous target cells. Virus-specific cytotoxicity required the presence of both IgG antibodies against measles virus and of effector lymphocytes. The effector lymphocytes had Fc receptors and were mainly present in a fraction of non-T lymphocytes. Monocytes were not cytotoxic but rather inhibitory. These results indicate that lysis of virus-infected cells in this direct assay is due to antibody-dependent cellular cytotoxicity (ADCC), caused by K cells. Control experiments showed that the virus-infected target cells were sensitive to incubation with human serum or IgG, resulting in a nonspecific increase of ⁵¹Cr release; however, this did not affect the results of K-cell cytotoxicity. Maximal virus-specific lysis by ADCC did not reach the level obtained by complement-dependent cytotoxicity. Possible explanations for this difference are discussed.