The cytotoxic effects of bee venom and melittin on cultured MCF7 cells were evaluated using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc., Gaithersburg, MD, USA). The cells were seeded at a density of 5×103 cells/200 µl/well into wells of 96-well round-bottomed plates and allowed to grow for 24 h at 37°C, followed by incubation with bee venom or purified synthetic melittin for 6 h at 37°C. The culture supernatants (100 µl quantities) were harvested and mixed with 10 µl aliquots of CCK-8 solution. Following 3 h incubation at 37°C, the optical densities at A450 were measured using the VICTOR™ X3 ELISA reader (PerkinElmer, Inc.).
Victor x3 elisa reader
Manufactured by PerkinElmer
Sourced in United States
The VICTOR™ X3 ELISA reader is a compact, automated, and versatile microplate reader designed for performing Enzyme-Linked Immunosorbent Assays (ELISA). It provides precise and reliable absorbance measurements across a wide range of wavelengths to support various ELISA-based applications.
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4 protocols using victor x3 elisa reader
1
Cytotoxic Effects of Bee Venom and Melittin on MCF7 Cells
2
Microdilution Broth Assay for Antimicrobial MIC
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To determine the minimum inhibitory concentration (MIC), the present study used a micro-dilution broth method, according to the recommendations of the National Committee for Clinical Laboratory Standards (19 ). In brief, the cells of the experimental bacterial strains were collected in the logarithmic phase of growth, suspended in 30 mM phosphate buffer (pH 7.0) with 60 mM NaCl, and adjusted to an A600 of 0.3 arbitrary units (1×105 cells/ml). The bee venom and the melittin samples were dissolved in 10 mM phosphate buffer (pH 6.0) with 130 mM NaCl and 0.2% (w/v) bovine serum albumin prior to serial dilution. Sample aliquots (10 µl) were mixed with the diluted bacterial suspensions (190 µl) followed by incubation for 20 h at 37°C. Bacterial growth was determined by measurement of the A650 levels using a VICTOR™ X3 ELISA reader (PerkinElmer, Inc., Waltham MA, USA).
3
Antibody Isotype Quantification by ELISA
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The antibody isotypes IgG, IgG1, and IgG2a titers were measured from peripheral blood using an indirect Enzyme-Linked Immunosorbent Assay (ELISA). Serum was collected at days 0, 15, 30 and 45 post-immunization. Ninety-six-well polystyrene microtiter plates (Thermo Fisher Scientific Inc., MA) were coated with 1 μg/ml recombinant SH3-267 or ZM270 proteins in 0.05 M carbonate-bicarbonate buffer (pH 9.6) and left overnight at 4°C in a humid chamber. After washing the plates with PBS-0.05% Tween-20 (v/v) buffer, they were treated with 0.8% gelatin (w/v) in PBS-Tween-20 buffer for 1 h at 37°C in order to block non-specific sites. Serial two-fold dilutions of sera containing primary antibodies from test and control animals were added and incubated for 3 h at room temperature. Rabbit anti-mouse IgG, IgG1, and IgG2a secondary antibodies conjugated with horseradish peroxidase (US Biological, Life Sciences) at a dilution of 1:1000, were added and incubated for 45 min. The reaction was revealed using OPD peroxidase substrate (Sigma-Aldrich Co., MO) and stopped with 50 μl H2SO4 2N. Results were read using a VictorX3 ELISA reader (PerkinElmer Chile LTDA, Santiago, Chile) at 490 nm. All assays were done in triplicate.
4
Cytokine Production in Infected Macrophages
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The contribution of the ORF BAB1_0270 to the attenuation of the mutant strain in the stimulation of infected macrophages was assessed through the production of the pro-inflammatory cytokines TNF-α and IL-6 by ELISA. For this, 5 × 105 RAW264.7 macrophages/well were seeded in 24-well plates (Nunclon Delta, Denmark) and infected with wt, mutant and inactivated strains at MOI 1:10 under the culture conditions described above. The production of these cytokines was quantified in the supernatants from macrophages infected 6 h and 24 h pi. TNF-α and IL-6 production was evaluated by ELISA using the commercial kits eBioscience Mouse TNF alpha and Mouse IL-6 ELISA Ready-SET-Go! (Fisher Scientific, MA, United States). Final concentrations of cytokines were quantified by standard curves based on the concentration of recombinant mouse TNF-α and IL-6. Results were obtained using a VictorX3 ELISA reader (PerkinElmer, Waltham, MA, United States) at 450 nm. All assays were done in triplicate.
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