Spectramax 190 reader

Enzyme immunoassay plates were coated with 50 µM/well of poly-lysine (PL) at 5 μg/mL then stored at 4 °C for 16 h. Aptamers were immobilized on wells and the plate was incubated overnight at 4 °C. To remove unbound aptamers, wells were washed three times with washing buffer (10 mM PBS, 0.05% Tween-20, pH 7.4,). The plate was blocked with 300 μL/well of a blocking agent (10 mM PBS, 1% BSA, pH 7.4) for 1 h at 37 °C; and another cycle of washes followed and 100 μL/well of different concentrations of virus in 10-fold serial dilutions (5,000,000–0 PFU/mL) was added. After incubation for 1 h at 37 °C, the unbound virus was removed by washing three times. One hundred microliters of monoclonal antibody 4G2 in a 10-fold dilution was added and the plate was incubated at 37 °C for another 1 h. Subsequently, the plate was washed again, 100 µL of anti-mouse conjugated with peroxidase at a dilution of 1/30,000 in PBS was added and the plate was incubated for 1 h at 37 °C. The plate was again washed three times and 100 µL of tetramethylbenzidine (TMB) chromogenic solution was added before 20 min of incubation at 37 °C. The color reaction was stopped by adding 100 µL of 1 N HCl and the absorbance was read at 450 nm using the Spectra Max190 reader (Molecular Devices, San Jose, CA, USA) supplied with the Soft Max Pro 6.3 software.

Citrate synthase activity was assessed as described by Hansford and Castro (1982) (link). Reactions were performed in a 96-well plate format containing 200 μL reaction buffer and substrates [Tris-HCl 0.1 M, pH 8.1, acetyl-CoA 5 mM, DTNB 100 μM, oxaloacetate 250 μM Triton X-100 0.1% (w/v)]. Reaction started after adding 2 μL of whole cell extracts adjusted to 1.5 mg/mL protein concentration (3 μg/well of protein) and absorption kinetics was measure at 412 nm in a SpectraMax 190 reader (Molecular Devices©) with one reading every 19 s (15 s for acquisition, 3 s for shaking and 1 s for waiting) for 5 min at 30 °C. Enzymatic activity was estimated as formation of DNTB-CoA from CoA-SH released after acetyl-CoA and oxaloacetate condensation. Values were calculated from TNB molar extinction coefficient (ε=13.6×10⁻⁶ M⁻¹×cm⁻¹ ou 13.6 μM⁻¹×cm⁻¹) and adjusted to represent the amount of citrate formation in mol per min per mg of protein (nmol/min/mg).

BT474 cells, a HER2-overexpressing human breast cancer cell line, were seeded at 5000 cells/well in a 96-well culture plate (Costar, Cat#3599) supplemented with RPMI-1640+10% FBS, and incubated overnight at 37 °C with 5% CO₂. The next day, the cells were incubated with serially diluted anti-HER2×PD1 BsAb or trastuzumab ranging from 8 pM to 150 nM in a final volume of 200 μL/well. The plates were incubated at 37 °C for 6–7 days and viability was quantitated with Cell counting Kit-8 (Dojindo, Cat#CK04). The plates were then read on a SpectraMax 190 reader (Molecular Devices) at 450 nm.

Cell proliferation was detected using CCK-8 assay (Beyotime Institute of Biotechnology, Shanghai, China) and Real-time Cellular Analysis (RTCA assay; ACEA Biosciences, Inc.; Agilent Technologies, Inc.), as previously described [23 (link)–25 (link)]. For CCK-8 assay, cells were transfected with the plasmids and seeded in 96-well plate at a density of 5, 000–10, 000 cells/well. Cells were cultured for 24, 48, and 72 hours, followed by incubation with 10 μl CCK-8 reagent, the absorbance values at 450 nm were determined by a Spectra Max 190 reader (Molecular Devices, LLC). For RTCA assay, 5, 000–10, 000 cells were seeded into the E-plate. The data were recorded using xCELLLigence software 2.0 (ACEA Biosciences, Inc.; Agilent Technologies, Inc.) and analyzed using GraphPad Prism 5.0 (GraphPad Software, Inc.).