A colorimetric assay for quantitating bovine neutrophil bactericidal activity

A colorimetric assay was developed for quantitating bovine neutrophil bactericidal activity against Staphylococcus aureus. The procedure used the tetrazolium compound, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT). The assay was conducted by incubating antibody-opsonized S. aureus with neutrophils in microtiter plates for 1 h at a ratio of 10 bacteria per neutrophil. Neutrophils were then lysed with saponin. The MTT was added and samples were incubated for 10 min. Live S. aureus reduced MTT to purple formazan. Dead bacteria and lysed neutrophils did not react with MTT. Bacterially-reduced formazan was solubilized by adding isopropanol and formazan production was quantitated by measuring absorption at 560 nm. Absorption of formazan was directly related to viable bacteria cell number and was used to determine the number of S. aureus not killed by neutrophils. The percentage of bacteria killed by neutrophils was determined by extrapolation from a standard formazan curve that was derived by incubating MTT with known numbers of S. aureus. The colorimetric MTT assay detected suppressed bactericidal activity after in vitro treatment of bovine neutrophils with colchicine, cytochalasin B, or phorbol 12-myristate 13-acetate. In vitro treatment of neutrophils with low levels of recombinant bovine interferon gamma (rBoIFN-gamma) enhanced bactericidal activity, whereas high levels decreased activity. These results suggest the colorimetric MTT bactericidal assay is efficacious in detecting modulation of bovine neutrophil bactericidal activity. Furthermore, the MTT assay has many advantages over traditional bactericidal assays in that it is sensitive, inexpensive, requires less than 3 h to complete, and can analyze many neutrophil samples in a single day.     

Growth and infectivity assays of the Israeli vaccine strain of fowl poxvirus in chicken embryo fibroblasts

The Israeli vaccine strain of fowl poxvirus grows efficiently in chicken embryo fibroblasts but not in cell lines derived from monkey kidney or human fibroblasts. We developed two assays for the titration of the infectivity of this virus in secondary cultures of chicken embryo fibroblasts. The first is a focus assay, in which minimum essential medium and SeaKem ME agarose were used for the overlay media. Under these conditions, clear virus foci appeared after 5 days of incubation at 37 C. The second assay is a semiautomatic colorimetric test based on the ability of live cells in culture to reduce the yellow tetrazolium salt 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; thiazolyl blue) to its formazan derivative. The reagent was added to infected chicken embryo fibroblasts in 96-well plates 10 days after infection. The formazan formed during 2 hr was extracted with dimethyl sulfoxide, and its absorbance was read by an automatic microplate spectrophotometer. A good correlation of the infectivity titers of the virus was obtained by the two methods.     

An evaluation of the mitogenic reactivity of intestinal intraepithelial lymphocytes of chickens.

A colorimetric assay employing MTT (3-[4,5-dimethylthiazole-2-yl], 2-5-diphenyltetrazolium bromide) was used to determine the mitogenic response of intestinal intraepithelial lymphocytes (i-IELs) of chickens to T- and B-cell mitogens. Comparisons between mitogenic responses of i-IELs and peripheral blood lymphocytes (PBLs) were made to examine potential relationships. The results from this study indicated that T-cell mitogens, concanavalin A (Con A), and phytohemagglutinin-P (PHA-P) induced mitogenic stimulation in i-IELs. Although stimulation indexes of both i-IELs and PBLs were similar, the optical densities (ODs) of i-IEL cultures containing Con A or PHA-P were 20- to 50-fold lower than the ODs of PBL cultures containing the same mitogen. The lower conversion of MTT to formazan resulting in lower ODs in i-IEL cultures indicated a lower level of cellular activity in the i-IELs than in the PBLs. The mitogenic responses of both i-IELs and PBLs to Con A and PHA-P were dose dependent. The responsive concentration of Con A for i-IELs was within the range of 25-50 micrograms/ml, whereas the responsive concentration of PHA-P for i-IELs was 50 micrograms/ml. Three days of incubation was found to be adequate to induce a significant (P < 0.05) mitogenic response for both T-cell mitogens. Lipopolysaccharide was unable to induce a mitogenic response in i-IELs, which was attributed to the lack of B cells in the i-IEL population. This technique may prove useful in evaluating and studying the role of i-IELs in local cell-mediated immune responses of the gastrointestinal tract.     

Use of a soluble tetrazolium/formazan assay for chicken cells.

We evaluated a soluble tetrazolium/formazan assay using 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl ]-2H- tetrazolium hydroxide (XTT) for chicken cell growth. Fifty microliter of solution containing 1 mg/ml of XTT and 0.025 mM phenazine methosulfate was added to the cells in a well of 96-well microplate. After 4 hr incubation at 37 degrees C, the absorbance was measured at 490 nm. Under this condition, absorbances were well correlated with cell number of Marek's disease tumor cells and chicken embryo fibroblasts. Proliferation of chicken lymphocytes stimulated with mitogens was also effectively measured. The formazan of XTT is water-soluble and can be quantitated in culture medium without the necessity for extraction with organic solvents. Thus XTT assay is simple and useful for the quantity assay with chicken cells.     

Evaluation of canine lymphocyte proliferation: comparison of three different colorimetric methods with the 3H-thymidine incorporation assay.

To evaluate canine lymphocyte stimulation the radioactive thymidine incorporation assay is still the method of choice. In order to find a suitable non-radioactive alternative to the standard 3H-thymidine incorporation assay, proliferation of canine peripheral blood lymphocytes (PBL) was measured with three different colorimetric assays, using the two tetrazolium salts MTT and XTT and 5-bromo-deoxyuridine (BrdU). Isolated canine PBL were stimulated with two different mitogens, Concanavalin A (Con A) and Phytohemagglutinin (PHA), using different culture conditions. Applying statistical analysis we found that BrdU and MTT showed a high correlation to the 3H-thymidine incorporation assay, although the BrdU assay proved to be more sensitive than the MTT assay. No significant correlation between the XTT assay and the radioactive method was demonstrated. Consequently, the BrdU assay is the most suitable alternative to the radioactive method.     

Comparison of MTT colorimetric assay and tritiated thymidine uptake for lymphocyte proliferation assays using chicken splenocytes.

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) colorimetric assay was compared with the conventional tritiated thymidine deoxyriboside (3H-TdR) incorporation for assay of lymphocyte blastogenesis using mononuclear cells isolated from the spleens of specific-pathogen-free chickens. The study was undertaken in an effort to simplify methods for assessing avian lymphocyte proliferation, specifically for evaluating response to mitogens or for indirect measurement of T-cell growth factors. The results from stimulated cells in both assay methods were significantly different from results from the control cells, and the MTT assay results regressed in a significant linear manner on counts from 3H-TdR incorporation. On this basis, the MTT assay is a valid test for evaluation of lymphocyte proliferation of chicken splenocytes.     

A colorimetric assay to evaluate the cytotoxic activity of the intestinal intraepithelial lymphocytes of chickens

After the successful use of 3-[4,5-(dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide (MTT) in cell proliferation assays, its use has been established by different workers in cytotoxicity assays and research on leukaemia. In the present study, a colorimetric assay using MTT was adopted to evaluate the cytotoxic activity of chicken intestinal intraepithelial lymphocytes (iIELs), which constitute an important cellular component of the gut-associated lymphoid tissue (GALT). These iIELs are found to exhibit natural killer (NK) cell-like cytotoxic activity, which is spontaneous, non-MHC-restricted, and does not need to be primed. Hitherto, conventional chromium-release assays have been used to evaluate the cytotoxic activity of iIELs, but these assays have disadvantages such as radiation hazards and loss of the cells in washing steps. The mean percentage cytotoxic activity of chicken iIELs evaluated by the colorimetric assay was 90.37±2.53 in a group of 5-week-old chickens and 80.2±3.45 in a group of 8-week-old chickens. These findings established the successful use of a colorimetric assay using MTT for evaluating the cytotoxic activity of chickens iIELs.Abbreviations DMEM Dulbecco's modified Eagle's medium - DMSO dimethyl sulphoxide - E effector cells - GALT gut-associated lymphoid tissue - GM growth medium - iIELs intestinal intraepithelial lymphocytes - MTT 3-[4,5-(dimethylthiazol-2-yl)]-2,5-diphenyltetrazolium bromide - NK cell natural killer cell - OD optical density - RPMI Rosewell Park Memorial Institute medium - T target cells     

以MTT比色法检测鸡脾淋巴细胞转化效果

本研究运用ＭＴＴ比色法以正交试验对鸡脾脏淋巴细胞转化的最佳条件（ｃｏｎＡ量、血清种类和浓度）进行了研究。结果表明，无论血清种类和浓度如何，低剂量ＣｏｎＡ（２．５～１０μｇ／ｍｌ）可以获得较好的转化效果。当ＣｏｎＡ的量确定时，低浓度鸡血清（０．５％～１．２５％）的转化效果优于高浓度（２％～３％）：５％犊牛血清优于任何浓度的鸡血清；无血清培养基优于血清培养基。其最佳组合为无血清ＲＰＭＩ１６４０培养基加ＣｏｎＡ２．５μｇ／ｍｌ。     

A rapid MTT colorimetric assay to assess the proliferative index of two Indian strains of Theileria annulata

A study was undertaken to compare the proliferative index of macroschizont-infected lymphoblastoid cells of two Indian strains [Izatnagar (IZT) and Parbhani (PBN)] of Theileria annulata by an in vitro MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide], colorimetric assay. Culture conditions were standardized to define the optimal cell concentration in 96-well microculture plates to yield nearly 100% living cells for measurement of the metabolized formazan activity. A cell concentration of 1.5x10(5) cells/ml was found to be optimal for effective discrimination of the parasite strains. On the basis of conversion of MTT by the actively proliferating lymphoblastoid cells, the PBN strain of T. annulata stimulated a 2.5-fold increase in formazan activity in comparison to the IZT strain. The in vitro MTT assay was found to be a simple and convenient method for assessing the cell activation rate and growth, obviating the need for radioactive material for the assay. The results of the proliferation assay are discussed in relation to previously documented information on the biological characteristics of this important pathogen of cattle.     

Adaptation of a colorimetric microtitration assay for quantifying Pasteurella haemolytica A1 leukotoxin and antileukotoxin

A colorimetric microtitration assay was adapted to quantify the cytotoxicity of Pasteurella haemolytica A1 leukotoxin to bovine neutrophils used as target cells. The viability of leukotoxin-treated target cells was detected by use of a tetrazolium dye that living cells reduced to dark blue formazan. The amount of formazan formed (which was quantified by use of an ELISA plate reader) was directly proportional to the number of viable target cells. This assay system also was used to measure leukotoxin-neutralization antibody titers of bovine serum and lung lavage specimens obtained during vaccination experiments. The major advantages of this assay over other methods such as the 51Cr-release and trypan blue-exclusion assays are precision, rapidity, and low cost; it also does not use radioisotopes.     

Evaluation of a nonradioactive colorimetric assay for analysis of lymphocyte proliferation in healthy cats

OBJECTIVES: To compare results of a nonradioactive colorimetric microplate assay with results of a traditional radioactive proliferation assay for determination of its use as a reliable and accurate alternative method for determination of proliferative activity of feline lymphocytes. SAMPLE POPULATION: Blood samples from 10 clinically normal domestic shorthair cats. PROCEDURE: Double-density gradient separation was used to isolate mononuclear cells. Isolated cells were stimulated with various concentrations of concanavalin A (Con-A) and cultured for 72 hours. Lymphocyte proliferation was measured by radioactive ([3H]thymidine) and nonradioactive (colorimetric) techniques. Immunophenotypic analysis with feline-specific CD4+ and CD8+ monoclonal antibody was performed, using flow cytometry. RESULTS: Mononuclear cells were successfully isolated (97 to 99% purity and viability) from blood samples. A similar dose-dependent proliferative response to Con-A stimulation was measured with [3H]thymidine incorporation and the colorimetric assay. For both techniques, concentrations of 0.1 and 1.0 microg of Con-A/ml were submitogenic, and 100 microg/ml was toxic to cultured cells. For both techniques, maximal proliferation was observed with 5 microg of Con-A/ml. CONCLUSIONS AND CLINICAL RELEVANCE: These results indicate that the nonradioactive colorimetric technique is a reliable and accurate method for measuring proliferative activity of feline lymphocytes. Clinically, this assay can be used as part of a screening process to determine immunocompetence of at-risk cats and to evaluate treatments for cats with immune-mediated or T-cell-dependent diseases.     

Canine lymphocyte cultures in vitro: Evaluation of peripheral blood lymphocyte response to mitogens

Lymphocytes from dog peripheral blood have been stimulated in vitro with 3 different mitogens (Con A, PHA and PWM). Culture medium was RPMI 1640 enriched with either autologous plasma, fetal calf serum or a newly described defined serum substitute. In such cultures the number of surviving and activated cells was measured by cytofluorometry and the proliferation was assessed by thymidine incorporation. In unstimulated cultures, up to 70% of all cells had disappeared (died) during the first 42 hours of incubation, whereas the number of viable cells was reduced to 50–60% in mitogen stimulated cultures. Of the surviving lymphocytes, between 25–40% of the cells appeared to have an elevated RNA-content (activated or G₁ cells). By comparison between thymidine incorporation and number of mitogen induced G₁ cells, a very high correlation was found (r=0.92). However, the slope of the regression line was much lower than expected. The low thymidine incorporation per activated cell was primarily related to the high cell death and a resulting dilution of tritiated thymidine. Indeed, preliminary results suggested that the same thymidine incorporation per G_1b cells could be obtained if peripheral blood lymphocytes were washed immediately before pulsing as could be obtained with lymphnode cells without washing.     

Stimulatory effect of avian influenza virus on chicken lymphocytes

A study was conducted to examine the effect of avian influenza virus (AIV) on chicken lymphocyte activation. Unprimed or Brucella abortus antigen (Ag)-primed lymphocytes were incubated with various doses of the T-cell mitogen concanavalin A (Con A) or Ag, respectively, plus serial dilutions of inactivated AIV for 72 hr, and cell proliferation was measured via uptake of tritiated thymidine. AIV enhanced the proliferative response to Con A or Ag by 150% or better, and the enhancement decreased in a viral dose-dependent manner. The effects were more readily observed in cells that had not been maximally activated by the Con A or Ag. The enhanced response was observed in lymphocytes from both white rock and white leghorn breeds of chicken and in mature peripheral blood lymphocytes or immature thymocytes. The viral activity could be abrogated by pre-treatment of the viral preparation with AIV-specific antisera or prior adsorption of the AIV with chicken erythrocytes. These results indicate that AIV can interact with and modify the in vitro activity of chicken lymphocytes and may exert modulatory effects on the avian immune system.     

Optimum conditions for the chicken lymphocyte transformation test.

Optimum conditions for chicken (Gallus gallus) lymphocyte transformation tests were determined. Thrice-washed chicken buffy-coat cells obtained after slow centrifugation (40 x g for 10 minutes) responded substantially better to mitogenic stimulation than lymphocytes isolated on separation media containing Ficoll. Maximum responses were obtained with 2 x 10(7) lymphoid cells/ml. Responses to the mitogens were greatest when fetal bovine serum was used at a 5% concentration or pooled chicken serum and autologous plasma were used at a 1.25% concentration. Optimum mitogen concentrations varied with individual birds, timing of the culture, temperature of incubation, and serum concentration in the cultures. When 1.25% chicken serum was used in the cultures, responses were usually greatest with final concentrations of 30-50 micrograms/ml of concanavalin A (Con A) and 30-50 micrograms/ml of phytohemagglutinin-P (PHA-P). The optimum concentration of pokeweed mitogen (PWM) varied from 1 to 40 micrograms/ml among the birds and was practically impossible to establish in general. The incubation in humidified air with 5% CO2 was significantly better at 40 C than at 37 C. The total culture time of 40 hours including pulsing with 3H-thymidine during the final 16 hours of incubation was the best for Con A- and PHA-P-stimulated cells, whereas a longer incubation of 64 hours gave the highest results with PWM stimulations.     

Lymphoblastic transformation assay of sheep peripheral blood lymphocytes: A new rapid and easy-to-read technique

A new micro-method was used to evaluate in vitro sensitivity of ovine peripheral blood lymphocytes (PBL) to different non specific mitogens (pHA, Con A, PWM) and to investigate the interest of a colorimetric assay for measurement of transformed lymphocytes.

The results showed that sheep PBL in flat-bottomed microplates responded optimally at a cell density of 8 × 10⁶ cells/ml to PHA (2.5 μg/ml), Con A (5 μg/ml) and PWM (5 μg/ml).

The colorimetric assay using a tetrazolium salt (MTT), for measuring the transformed lymphocytes, is very well correlated with the classical method of [³H]thymidine incorporation.

This new revelation technique of the mitogenic response improve the technical value of the assay, which is more rapid and easy-to-read, without diminishing the biological value.     

On the suitability of the MTT-assay for the evaluation of mitogenic lymphocyte blastogenesis in swine

Using swine peripheral blood lymphocytes, we examined the suitability of a colorimetric tetrazolium (MTT) reduction test for the evaluation of mitogenic lymphoblastogenesis in comparison to radiolabelling methods. In our study, we did not find a correlation between the 3H-thymidine- or 14C-thymidine uptake into DNA and the mitochondrial MTT cleavage activity, comparable to that formerly demonstrated for mouse cytotoxic T cells and for sheep peripheral blood lymphocytes. With the MTT assay, mitogen-driven lymphocyte activation was scarcely assessable, whereas a clear mitogenic response could be observed using radiolabelling methods. The results show that the MTT cleavage activity reflects quite a different state of cellular function than the DNA synthesis and that the evaluation of this activity is not suited to determining lymphocyte activation in swine as a measure of mitogenic response.     

Phytomitogen- and antigen-induced blast transformation of feline lymphocytes.

A semiautomated microassay of blast transformation of feline lymphocytes was developed and used in a study of the response of normal cats to phytomitogen- and antigen-induced stimulation. Peripheral blood lymphocytes were isolated from defibrinated blood by ficolldiatrizoate gradient centrifugation and incubated at a concentration of 1 X 10(5) cells/culture. Blast transformation was measured by the incorporation of tritiated thymidine, to which the cells were exposed during the last 18 hours of incubation. Phytomitogens induced maximal transformation after 3 days' incubation. Greatest stimulation produced by 10 mug of concanavalin A (38- to 183-fold) followed by 4 mug of pokeweek mitogen (nine- to 51-fold), and finally phytohemagglutinin-P (three- to sixfold). Three of 4 cats sensitized to keyhole limpet hemocyanin responded when exposed to 50 mug of keyhole limpet hemocyanin in vitro, maximal transformation occurring after 4 days' incubation. Lymphocyte blast tranformation provides a convenient technique to assess lymphocyte function serially during experimentally induced immunologic alterations in the cat.     

Culture conditions for blastogenic responses of bovine mammary mononuclear cells

Several experimental parameters were examined to determine optimal conditions for proliferative responses of mammary mononuclear cells (MMC) obtained from six nonlactating dairy cows. These parameters were: pre-incubation of cells in medium prior to assay, mitogen concentration, assay incubation time, and type of culture medium. Response variables included viability of cells and the rate of proliferation as assessed by tritiated thymidine incorporation. Pre-incubation of cells in medium had no effect on the proliferative response of MMC. Whereas Concanavalin A (ConA; 3.3 or 6.6 micrograms/ml) and phytohemagglutinin (PHA; 1, 5, 10 micrograms/ml) did stimulate proliferation of MMC, the higher doses did not stimulate greater proliferation than the lower doses of mitogens. The greatest mitogenic response was obtained on days 2 and 3 of incubation. Proliferative responses were significantly higher at all mitogen levels tested in a 50-50 mixture of Rosewell Park Memorial Institute medium 1640 and Liebovitz-15 medium (RPMI/L-15) than in RPMI alone. Viability of MMC was also significantly higher in the RPMI/L-15 medium. To test whether the significant effect of media on blastogenesis was specific for mononuclear cells from the bovine mammary gland, peripheral blood lymphocytes (PBL) from four dairy cows were cultured with ConA and PHA in a mitogen assay in both RPMI and RPMI/L-15. Viability was measured on day of collection and on all culture days. PBL were stimulated equally in both media. PBL viability decreased significantly on day 1 in both RPMI and RPMI/L-15. These results suggest that the optimal culture conditions for blastogenic responses of mammary mononuclear cells and peripheral blood lymphocytes may differ.     

Immunomodulatory properties of a strain of Mycobacterium chelonae. I. Mouse lymphocyte responses in vitro

Immunomodulatory properties of a strain of live Mycobacterium chelonae (Mch) was investigated in an in vitro lymphocyte transformation system. Murine splenocyte activation by this bacterium was characterized by polyclonal lymphoproliferative responses in a dose dependent fashion. Optimal doses ranging from 20 to 80 micrograms of Mch (wet weight) per ml of cell suspension induced a very significant mitogenic effect. Higher doses (100 micrograms) of Mch manifested a decreased rate of tritiated thymidine ([3H] TdR) uptake whereas responsiveness of splenic lymphocytes to lower doses (0.156 microgram) was not modified. Contrary to the splenocyte responses activation of murine thymocytes by this mycobacterium is characterised by a decreased proliferation as compared to the background count of unstimulated cells. Simultaneous addition of Mch with optimal doses of Concanavalin A (Con A) and Phytohemaglutinin (PHA) potentiated polyclonal mitogenic responses of murine splenocytes to these two lectins. However, proliferation of these lymphocytes to Lypopolysaccharide (LPS) induction was not modified. BALB/C and DBA/2 spenocytes were found to be more responsive to stimulation by this Mycobacterium as compared to those of C3H/Ou and to a lesser degree to those of C57BL/6 mice.     

Rapid, multiwell colorimetric assay for measuring neutrophil chemoattractant activity in bronchoalveolar lavage fluid of horses with recurrent airway obstruction.

The criteria used to diagnose recurrent airway obstruction (RAO) in affected horses include demonstration of reversible lower airway obstruction and greater than 25% neutrophils in bronchoalveolar lavage fluid (BALF). Additional objective laboratory tests are needed to improve diagnostic accuracy and to monitor response to treatment. The goal of this study was to determine if neutrophil chemoattractant activity of BALF could be measured by using a previously described, rapid, multiwell colorimetric assay for chemotaxis. In this assay, neutrophils that have migrated through a membrane filter are collected into the bottom well of a disposable chemotaxis-cell migration chamber. The number of viable cells collected in the bottom well is quantified by measurement of the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide (MTT), which is reduced by dehydrogenase in mitochondria of live cells. The number of migrating cells corresponds to the amount of MTT reduced, which is measured with an enzyme-linked immunosorbent assay plate reader. Fourteen adult horses were enrolled in this study, 7 of which had owner histories consistent with RAO. Each horse was sedated, a bronchoalveolar lavage tube was passed, and saline was infused and immediately aspirated. An aliquot of BALF was used for differential cell count, and BALF supernatant was harvested to assess neutrophil chemoattractant activity. Normal control horses and RAO-affected horses were distinguished according to clinical signs and percent neutrophils in BALF. Neutrophil chemoattractant activity of BALF was significantly greater in RAO-affected horses (P = 0.001) compared with control horses. This assay may be useful in future studies for monitoring response to therapy in RAOaffected horses.