Coelenterazine

ELISA plates were coated overnight at 4 °C with 100 µL 1 µg mL⁻¹ neutravidin in PBS. Plates were washed three times with PBS and then blocked by filling to brimming with Bb for 1 h. A volume of 100 µL 100 nM anti-MARV sdAb A, B or C as BAP fusion proteins purified from pecan126 as described above was applied to duplicate wells in Bb for 1 h. Wells were washed to brimming three times with PBST and two times with PBS. Bb was added to the well to brimming for 1 h to further block the sdAb and then dilutions of nluc, nluc–MARV–NP600 or nluc–MLAV–NP604 in MPBS were added to duplicate wells for 1 h. Following washing, wells were developed with injection of coelenterazine (NanoLight™ Technology, Pinetop, AZ, USA) in lucky buffer (10 mM Tris, 1 mM EDTA, 500 mM NaCl, pH 7.4) and emissions collected using a luminometer (Turner Biosystems, Sunnyvale, CA, USA) with a 2 s integration. The experiment was repeated two more times and curves are the plots of three mean RLU of nluc–NP minus the corresponding mean of the nluc alone with error bars representing +/− SD. The EC₅₀y value was calculated for curves that plateaued using the equation [RLU_min + (RLU_max − RLU_min)/2]. The corresponding x values were calculated using one observed point greater and one less than the y EC₅₀ using the trend function in Excel and the three values averaged and presented +/− SD nM.

The ΔluxCDABEG strain (parent) and its ΔcasA derivative carrying pAT103 were grown overnight at 28°C in LBS with Cm. The overnight cultures were subcultured 1:100, grown until mid-log phase, and collected and centrifuged. Samples were washed two times and resuspended in PBS, and a limiting amount of coelenterazine (Nanolight Technology) was added to a final concentration of 5 μM. The samples were vortexed and incubated in the dark for 1 h and washed in PBS, and the optical density at 600 nm (OD₆₀₀) was measured. Samples were normalized to an OD of 0.4 in a white 96-well plate and incubated in the dark for 10 min. The baseline luminescence of the samples was measured with a delay of 1 s for 5.9 min in a luminometer (Veritas microplate luminometer; Turner Biosystems). Calcium was added to a final concentration of 40 mM, and the luciferase activity was measured with a delay of 1 s for approximately 17 min 40 s. The limiting amount of coelenterazine is responsible for the temporal appearance of light production. This assay was performed at least three separate times.

Twenty-four hours after transfection, the CHO cells were washed in PBS and afterwards resuspended in PBS + 1% glucose. Eighty µl of 4 × 10⁶ cells/ml were seeded in a 96-well plate (~100,000 cells/well). Five µM coelenterazine (NanoLight Technology, Pinetop, AZ, United States) was added to the cells. After 10 min, 5 µl of varying ligand concentrations was added to each well. After 15 min coelenterazine and 1 µM forskolin (Sigma-Aldrich) were added. The plates were kept in the dark all the time. The emission signals from Renilla luciferase (RLuc) and yellow fluorescent protein (YFP) were measured with a 2104 EnVision Multilabel plate reader (PerkinElmer, Waltham, MA, United States). The BRET signal is the ratio of the detected YFP (acceptor emission) at 525 nm divided by the RLuc at 475 nm (donor emission) [30 (link)].

K562 (Chronic Myelogenous Leukemia), Raji (Burkitt’s lymphoma), HEL 92.1.7 (Erythroleukemia) and NK92MI cell lines were obtained from ATCC and maintained as per the instructions provided. Nalm6 (Acute Lymphoblastic Leukemia), BC-1 (Primary effusion lymphoma), and KG-1 (Acute myelogenous leukemia) cell lines were kindly provided by Drs. Markus Muschen, Jae Jung, and Alan Epstein, respectively. 293FT cells were obtained from Invitrogen (ThermoFisher Scientific) and cultured as recommended. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient method from platelet depleted donor cells obtained from a Blood Bank. PBMCs were subsequently used to isolate T cells using CD3 magnetic microbeads (Miltenyi Biotech) following the manufacturer’s instructions. T cells were cultured in XVIVO-15 (Lonza) medium supplemented with 100 IU/ml recombinant human IL2 and 30 ng/ml soluble antibodies to human CD3 and CD28. All the cells were cultured at 37 °C, in a 5% CO2 humidified incubator. Blinatumomab was obtained from Amgen, Rituximab was obtained from Genentech. Digitonin, and Polybrene were from Sigma. Coelenterazine was purchased from Nanolight technology. Calcein AM fluorescent dye was obtained from BD biosciences.

eGFP expression was visualized using a microscope with a fluorescent light source (Leica MZ10F). fluc expression was quantified by non-invasive bioluminescence imaging (IVIS Spectrum, Perkin Elmer) at the molecular imaging for Small Animals Center (moSAIC) at KU Leuven. Mice were sedated with isoflurane inhalation and subcutaneously injected with 126 mg/kg D-luciferin substrate (Promega) at 15 mg/ml in sterile D-PBS (14190144, Thermo Fisher Scientific). Total radiance (p/s, photons per second) was collected at fluc signal plateau. Gluc in plasma was analyzed using a FLUOstar Omega Microplate Reader (BMG Labtech) in black 96-well plates. Per well, 100 µl of 100 µM coelenterazine (Nanolight Technology, Pinetop, AZ) was added to 5 µl plasma via automated injection. Data was analyzed with MARS Software (BMG Labtech). Counts (RLU, relative light unit) 1 sec after injection of the substrate were used for analyses. Readout was corrected for the pretreatment plasma count.