A rapid method for detection of cellular proliferation using carboxyfluorescein Assay of growth factors (IL-2, IL-1) and growth inhibiting antibodies

The uptake of carboxyfluorescein diacetate (CFDA) into live cells was used as the basis for a simple, rapid and fully automated micromethod for determination of cell growth. The aim of the investigation was to adapt the CFDA method for detection of cell growth factors from cell culture supernatants. Thus, the biological activities of the growth factors IL-2 or IL-1 could be detected and quantitated at the same level of sensitivity as with the conventional [³H]thymidine incorporation. Similarly, the method was well suited to assay inhibition of IL-2-dependent lymphocyte growth by the monoclonal antibody anti-Tac, binding to the human lymphocyte membrane receptor for IL-2. The CFDA method proved to be rapid, reliable and well suited for several applications involving limiting numbers of cells and biological reagents.     

A sensitive rosetting method for detecting subpopulations of lymphocytes which react with alloantisera

A procedure is described for directly estimating the proportion of mouse lymphoid cell suspensions which react with alloantisera. The method entails reacting Ig-capped lymphoid cells with alloantisera, and then assessing the uptake of alloantibodies by rosetting the lymphocytes with SRBC coated with sheep IgG specific for mouse Ig. This rosetting procedure was found to be generally more sensitive than the conventional dye exclusion microcytotoxicity test for detecting the binding of alloantibodies to lymphocytes. Furthermore, the rosette method has the advantage that, unlike the complement lysis technique, it has a low and reproducible background and lymphocyte subpopulations which react with alloantisera can be isolated.     

活体染料CFDA-SE在淋巴细胞增殖研究中的应用

目的 :探讨活体染料CFDA SE在淋巴细胞增殖研究中的应用价值。方法 :利用CFDA SE染色、荧光抗体标记和流式细胞术 ,检测淋巴细胞及其亚群在多克隆刺激剂作用下荧光强度的变化 ,并应用相关软件分析其增殖情况。结果 :PDB ion omycin或ConA刺激 4 8h后均出现淋巴细胞分裂 ,表现为CFSE荧光强度的系列减半。环孢菌素A(CsA)可抑制ConA促进淋巴细胞增殖的作用 ,CFSE荧光无减半。ConA刺激4 8h后 ,出现CD4 T细胞和CD8 T细胞增殖不同步的现象 ,72h后这一现象更加明显。经ModFitTM 软件拟合后 ,得到的各项增殖指标显示 ,ConA对CD8 T细胞的促增殖作用强于对CD4 T细胞的作用。结论 :CFDA SE染色结合荧光抗体标记和流式细胞术 ,是分析淋巴细胞增殖的有力工具     

Enzymatic modification of the lymphocyte surface

Lymphocytes were treated with hydrolytic enzymes primarily to assess whether such modified cells would give improved cytotoxicity reactions during tissue typing. Papain-treated and alpha-chymotrypsin-treated lymphocytes were approximately twice as sensitive as untreated cells in the microcytotoxicity test used, and this finding might be usefully exploited by immunological laboratories for purposes of cross-matching, HLA antibody screening and HLA-DR typing. Trypsin treatment promoted massive cell clumping, while neuraminidase treatment was responsible for indiscriminate cell death after exposure to rabbit serum. The capacity of lymphocytes to form rosettes with sheep erythrocytes was abolished after treatment with trypsin or alpha-chymotrypsin, but enhanced by papain or neuraminidase.     

Detection of Alloantibodies Using a Sensitive Antiglobulin Microcytotoxicity Test: Identification of Low Levels of Pre-Formed Antibodies in Accelerated Allograft Rejection

An antiglobulin test, more sensitive than established direct microcytotoxicity tests, was developed to detect both complement fixing and non-complement fixing alloantibodies. The test has been used to detect antibodies in sera from selected organ allograft recipients. Of 56 sera tested by the antiglobulin method, 52 (93%) were reactive. In contrast, only 13 (23%) were reactive in an established direct microcytotoxicity test and only 28 (50%) were reactive in a modification of the direct test in which the incubation times were doubled. Sera obtained prior to transplantation from 3 patients who experienced either immediate or early rejection of their renal allografts contained antibodies detected by the antiglobulin test but not by direct microcytotoxicity tests against lymphocytes from a panel of unrelated, normal individuals. In addition, the antiglobulin test has been used to demonstrate that the humoral antibody response following transplantation of cardiac and renal allografts is heterogeneous with respect to formation of antibodies of the IgA, IgG and IgM classes.     

Flow cytometry with crystal violet to detect intracytoplasmic fluorescence in viable human lymphocytes. Demonstration of antibody entering living cells

A new method is described for the detection of intracytoplasmic fluorescence and its differentiation from surface staining of viable human lymphocytes using flow cytometry after addition of crystal violet which quenches surface but not internal fluorescence. This has then been used to study antibody penetration of viable human lymphocytes, using FITC-conjugated IgG from normal serum or serum containing anti-RNP antibody. The results showed that 54 +/- 1% normal lymphocytes were penetrated by anti-RNP antibody and 23 +/- 3% by normal IgG respectively. The lymphocyte population analysed by flow cytometry has been directly demonstrated to be viable by FDA staining. These results provide unequivocal evidence that antibody can penetrate viable human lymphocytes.     

A new sensitive and rapid automated fluorometric assay for detection of natural killer activity using carboxyfluorescein diacetate

An automated fluorometric assay using carboxyfluorescein diacetate (CFDA) has been applied for the sensitive and rapid detection of natural killer (NK) activity. The lysis of target cells by NK cells was quantified by measuring the amount of CFDA released into the supernatant of culture wells with the aid of an automated microfluorometer. Both sensitivity and specificity of the presented method were higher than the 51Cr release assay. Moreover, the detection of human NK activity against K562 target cells required only 2 hrs, compared to 4 hrs in the standard 51Cr release assay.     

Characterization of a method using viable human target cells as the solid phase in a cell concentration fluorescence immunoassay (CCFIA) for screening of monoclonal antibodies and hybridoma supernatants

A new method has been characterized for the use of viable target cells as the solid phase for screening of hybridoma supernatants in a cell concentration fluorescence immunoassay (CCFIA). Briefly, the specific target antigen on the cells is bound by the monoclonal antibodies and revealed by use of a fluoresceinated second antibody. Separation of free from bound antibody is accomplished by filtration in the 0.2 micron filter-bottom wells of specialized assay plates. Processing is automated in a Pandex screen machine, resulting in numerical fluorescence values for each well. This method is rapid (under 1 h per 96-well plate), highly sensitive (down to 0.2 ng/ml) and sparing of target cells (0.3-2.5 X 10(4) cells per assay well). It has been applied to 37 different varieties of human solid tumor cells, as well as to human peripheral blood mononuclear cells. The cells used as targets for the characterization of this method were still capable of attachment and growth when recovered post-assay. This method was compared with a viable cell enzyme-linked immunosorbent assay (ELISA) method, showing similar sensitivity and greatly shortened assay time. Comparison of the results from this method with those obtained from flow cytometric analysis performed on viable cells showed close correlation, whereas a lower correlation was seen with immunohistochemical methods using acetone-fixed cells. Development of this method made it possible to rapidly screen many thousands of hybridoma supernatants and successfully select those which were specific for surface antigens on viable cells.     

Spontaneous release of cytotoxic alloantibody from viable cells sensitized in excess antibody

When viable cells sensitized in excess cytotoxic alloantibody are washed and resuspended in antibody-free medium, they spontaneously release appreciable quantities of antibody. The amount released is directly proportional to the concentration of alloantibody during sensitization. Spontaneous release was observed from all cell types tested (thymocytes, lymphocytes, leukaemia cells and ascites sarcoma cells) and with all alloantibodies tested (H-2, θ and Ly-B). In preliminary tests with radio-labelled H-2 antibody, the quantity of antibody released in a period of 2½ hours was 29 per cent of the antibody originally absorbed. Dissociation at 37° was greater (or more rapid) than at 1°. When washed sensitized cells were suspended in antibody directed to an antigen closely adjacent on the cell surface to the site of attachment of the first antibody, release of the first antibody was impeded.     

Critical appraisal of complement dependent microlymphocytotoxicity assay for detecting donor-specific alloantibody pretransplant—importance of indirect immunofluorescence as a superior alternative

The ability of complement-dependent microlymphocytotoxicity assay (CdL) to detect and discriminate between the various types of donor-specific alloantibodies was reevaluated. Data obtained with the CdL assay on purified B and T lymphocytes at warm and cold temperatures was compared to other modes of antibody detection, i.e., indirect immunofluorescence (IF) and the noncomplement-dependent antibody-dependent cellular cytotoxicity (ADCC). Additionally, the significance of antibodies as detected by CdL and IF was ascertained by corelating with kidney transplant outcome. It became apparent that the CdL assay identified weaklyreactive HLA- ABC alloantibodies as being B cell specific. Such weakly reactive HLA-ABC antibodies were also not appreciated in the presence of the cold reactive IgM antibody. Accelerated rejections were the rule in the presence of weakly reactive HLA-ABC alloantibodies indicating that their detection was highly important. The IF assay could discriminate between the antibody class. could detect weakly reactive HLA-ABC alloantibodies, and could detect noncomplement fixing antibodies (ADCC). Further, use of IF prevented us from unnecessarily denying transplants to certain recipients when a positive CdL assay resulted from an IgM antibody or poor cell viability.     

Detection of red cell antibodies: comparison of two low ionic strength diluents

Various low ionic strength diluents are used routinely for red cell alloantibody detection in the antiglobulin test to increase the rate of antibody association to antigen, thereby allowing a reduction in the incubation time while achieving optimal agglutination. Two commercial low ionic strength diluents (DiaMed ID-CellStab and Inverclyde LISS) were assessed using the DiaMed-ID LISS Coombs' microtube column system, to assess whether or not the choice of diluent influences red cell antibody detection. Effects of two low ionic strength diluents after 15-min incubation were assessed in 150 samples containing a wide range of typical red cell alloantibodies. Inverclyde LISS gave significantly higher reaction strengths in 25% of samples when compared with the same red cells suspended in ID-CellStab. Variation in reaction strengths ranged from 1+ to 2+, using Inverclyde LISS versus CellStab. Of 131 red cell alloantibodies directed against Rh, Kell, Kidd and Duffy antigens, Inverclyde LISS detected 90% after 15-min incubation, whereas 83% were detected with CellStab. This study suggests that Inverclyde LISS provides better red cell alloantibody detection than does ID-CellStab, and this may be due to the higher ionic strength of ID-CellStab.     

HLA-D and -DR antigens on human amniotic fluid cells. II. Heterogeneous expression of HLA-DR and other cell surface markers

To evaluate further the feasibility of HLA typing for prenatal diagnosis, we tested human amniotic fluid cells (AFC), known to express HLA-A, -B, and -C antigens, for the presence of HLA-DR antigens using type-specific antisera in the microcytotoxicity assay and a monoclonal antibody directed against the common HLA-DR structure (cDR) in indirect immunofluorescence. Prenatal typing of HLA-DR on AFC in the microcytotoxicity test was possible in only one out of eight families studied. The detected DR2 antigen was confirmed by postnatal typings of cord blood lymphocytes. Thereafter, 23 different AFC cultures were tested with monoclonal antibodies in indirect immunofluorescence. Only six cultures were partially positive (23-35% fluorescent cells) with the monoclonal cDR antibody while all AFC cultures demonstrated strong positive fluorescence (68-100%) with a monoclonal antibody against the common HLA-A, -B, and -C structure (cHLA). These data suggest that only a small subpopulation of AFC expresses class II (HLA-DR) antigens in contrast to the nearly ubiquitous expression of class I (HLA-A, -B, and -C) antigens. Furthermore, the heterogeneous expression of cell surface antigens within the various AFC cultures was substantiated with monoclonal antibodies directed toward cell surface antigens of the OKT, OKM, and Lyt series that have been found to be characteristic for subpopulations of lymphoid and hemapoetic cells. Thus, at present, HLA-DR typing is not reliable for prenatal diagnosis.     

Cell-mediated cytotoxicity. A highly sensitive and informative flow cytometric assay

Determination of target cell lysis by cytolytic effectors has typically been achieved by two methods: the release of various markers from the cell, as in 51chromium release assays and the uptake of markers into the cell, as in trypan blue uptake in single cell/conjugate binding assays. Problems associated with these assays might include: (1) poor uptake, (2) nonspecific release, (3) poor statistics, (4) length of assays, or (5) subjectivity. These difficulties prompted the development of a new sensitive flow cytometric assay employing two fluorochromes. PKH-1, a fluorochrome which fluoresces in the green, binds to the cytoplasmic membrane and does not leak or transfer, is used to identify the target cell population. Propidium iodide fluoresces in the red and is used to detect non-viable cells. Use of these two fluorochromes and two parameter analysis allows for identification of four subpopulations in the sample: live effectors, dead effectors, live targets and dead targets. By enumeration of these subpopulations the following information can be calculated: (1) the percent target lysis, (2) effector-to-target cell ratios, (3) viability of the effector cells at the termination of the assay, and (4) viable effector to target cell ratios. The results show that PKH-1 labeling of target cells had no effect on effector-target cell interactions. Excellent correlation was found between this method and the chromium assay, however, due to earlier detection of the lytic event, this method provides a distinct time advantage over current methods.     

Antibody-bearing liposomes as multicolor immunofluorescence markers for flow cytometry and imaging

Loposomes covalently coupled to monoclonal antibodies retain the specificity of the antibody and bind only to cells bearing the appropriate determinant. As opposed to directly labeled antibodies which generally have fluorochrome to protein ratio of between 2–5, the entrapped space inside liposome can contain several hundred to several thousand molecules of fluorochromes in a space chemically isolated from the outside environment, thus providing the potential for an amplified fluorescence signal. We have prepared small unilamellar liposomes containing the soluble fluorochromes carboxyfluorescein (CF), which fluoresces in the green and sulforhodamine (SR), which fluoresces in the red, and covalently coupled a series of monoclonal antibodies using a heterobifunctional reagent. We were able to detect, on an Epics 753 flow cytometer equipped with an argon ion and a dye laser and by fluorescence microscopy, both single and double labeled mouse spleen lymphocyte subsets, fibroblast L cells and Raji cells. Complete color separation was obtained with CF-labeled cells being detected only by the green photomultiplier and SR-labeled cells by the red photomultiplier. Cells labeled with both were detected by both photomultipliers. Liposomes bearing anti-Ia antibodies bound only to B lymphocytes whereas those with anti-H-2K antibody bound both to T and B lymphocytes. In another system, single and dual color immunofluorescence made possible the simultaneous detection of HLA and H-2K molecules on transfected murine fibroblast L cells. The signal-to-noise ratio was more favorable for the liposome-labeled reagents than reagents labeled with fluorescein isothiocyanate. Cells labeled with antibody-bearing liposomes could be fixed with paraformaldehyde or glutaraldehyde without adversely affecting the original staining patterns. Apart from the two fluorochromes described above, other markers of choice could be encapsulated without any adverse effect on the antibody-liposome coupling procedure or on the specificity of the conjugated antibody. Since the fluorochrome is not directly coupled to the protein, there is no requirement for protein conjugation sites in order for it be usefully encapsulated inside liposomes. Therefore, this system provides new opportunities to exploit different, as yet untapped fluorochromes for use in flow cytometry and imaging.     

Natural antibodies to human lymphocytes and erythrocytes in the serum of Orcinus orca killer whale

The existence of naturally occurring heterophile antibodies to antigenic determinants on human blood cell membranes has long been known. It has been shown that the serum of Orcinus orca (Killer whale) does contain similar antibody. Absorption techniques in concert with either microagglutination or complement-dependent microcytotoxicity assays revealed at least three antibody specificities erythrocyte (RBC), B-lymphocyte and T-lymphocyte. Human erythrocyte specificity has been separated from other mammalian RBC specificity, and higher microagglutination titers and/or scores were observed with human group A RBC's than with group B,O, or AB. Tests run at 4 degrees, 20 degrees and 37 degrees C). Higher microcytotoxicity and microagglutination activity was demonstrated with B versus T lymphocytes. It is hoped that the characterization of the antigenic specificity of these heterophile agglutinins will prove to be useful as a biological reagent-tool which may be applied to the identification of a new receptor on human lymphocytes and/or erythrocytes. Also, if isolated, these agglutinins could be useful in the study of the occurrence and presence of specific receptors on cell membranes and give insight as to how these receptors change in health, disease and malignancy.     

Double fluorescence technique for measurement of complement-fixing antibody to lymphocyte subsets

A double fluorescent antibody method for quantitating human complement-fixing antibody to lymphocyte subclasses has been developed. The indicators in this system are a C6-deficient serum as a non-lytic source of complement, rhodamine-labeled anti-C3 and fluorescein-labeled murine monoclonal antibodies to human lymphocyte subsets. The basic procedure is to incubate lymphocytes with the unknown serum and then to add C6-deficient serum. The binding of C3 is indicated by staining with rhodamine-labeled anti-C3 and the subset class of the lymphocyte so stained is determined by binding of fluorescein-tagged anti-OKT4 or -OKT8 antibodies. The occurrence of both red and green cell surface fluorescence denotes the presence of a complement-binding antibody to the lymphocyte subset defined by the monoclonal antibody. In addition to defining the specificity of complement-fixing anti-lymphocyte antibodies, this technique is more sensitive than the microcytotoxicity assay.     

Stimulating effect of an arteriovenous shunt on the in vivo growth of isografted fibroblasts: a preliminary report

Isogenous fibroblasts derived from the skin of inbred Sprague-Dawley rats were cultured in vitro, labeled with bisbenzamide (BB) or carboxyfluorescein diacetate (CFDA), and seeded into polycarbonate growth chambers. After 24 h incubation in vitro, the chambers, either empty or containing an arteriovenous (AV) shunt, were implanted subcutaneously into the inguinal region of Sprague-Dawley rats and examined by fluorescence microscopy 2 or 7 days later. The AV shunt remained patent in all experiments. The density of labeled cells on the chamber surface in all chambers decreased in the first 2 days after insertion. At 7 days, the cell density in the empty chambers had not altered from the 2-day level, but the density in the AV shunt containing chambers had increased to almost three times the day 2 level (p = 0.013). It appears that an AV shunt can induce a significant proliferation of fibroblasts implanted adjacent to it. For at least 7 days after labeling, BB and CFDA provide a simple and effective method of quantitative detection of implanted fibroblasts. It is concluded that nutrients from the AV shunt implanted in a growth chamber result in a significant increase in the number of viable, matrix-synthesizing cells, compared with AV shunt-free controls.     

The application of radiolabelled HL-A antibodies in the detection of (soluble) transplantation antigens: an alternative for the microcytotoxicity test.

The use of radiolabelled anti HL-A antibody preparations is described. Absorption by platelets, followed by acid elution, fractionation of the eluate by column chromatography, radiolabelling and a second absorption--elution step with platelets yielded specific preparations, irrespective of original cytotoxic titres. The binding of these radiolabelled antibodies to lymphocytes could be inhibited by soluble antigens present in normal plasma or obtained from spleen cells by papain treatment. As a method for the detection of soluble transplantation antigens the use of radiolabelled antibodies appeared as sensitive as the microcytotoxicity test.     

AchR或ConA诱导正常人淋巴细胞培养上清治疗EAMG的研究

观察乙酰胆碱受体 (AchR )或刀豆蛋白 (ConA )诱导的正常人淋巴细胞培养上清对小鼠实验性重症肌无力模型 (EAMG )的疗效。分别用AchR或ConA诱导体外培养的正常人淋巴细胞 ,收集培养上清治疗EAMG小鼠。治疗前后用ELISA和微量细胞毒试验检测小鼠血中AchRAb的含量和T细胞亚群的变化 ,并检测小鼠的肌电图。结果显示 ,经两种培养上清治疗后 ,EAMG小鼠血中AchRAb水平明显下降 ,CD8+ 细胞数量显著增多 ,CD4+ /CD8+ 比值明显降低 ,且小鼠肌电图也明显改善。本文提示AchR或ConA诱导的正常人淋巴细胞培养上清对EAMG小鼠有一定疗效 ,并探讨了其可能的作用机制和潜在的应用价值。     

Alloantibodies that react with subsets of human T cells

Using the two color fluorescence (TCF) method, alloantibodies against subsets of T cells could be detected in sera from pregnant women with strong HLA antibodies. To preclude interference of these HLA antibodies with the recognition of the T cell antibodies, serum donors were selected which were HLA-Al, -B8, -DRw3. Their sera were tested on a panel of individuals homozygous for HLA-Al, -B8, -DRw3. By enriching peripheral mononuclear blood lymphocytes for Tgamma cells it could be shown that some of the sera reacted mainly with Tgamma and others with Tmu lymphocytes, while some sera reacted with both.