Power scan ht

ADCC induction by EpMab-37-mG_2a-f was assayed as follows. Six female BALB/c nude mice (five-week-old) were purchased from Charles River Laboratories, Inc. Spleens were aseptically removed, and single-cell suspensions were obtained through a sterile cell strainer (352360, BD Falcon). Erythrocytes were removed with treatment of ice-cold distilled water. The splenocytes were resuspended in the medium; this preparation was designated as effector cells. Target cells (BT-474 and Capan-2) were treated with Calcein AM (10 μg/mL, Thermo Fisher Scientific, Inc.). The target cells (2 × 10⁴ cells) were mixed with effector cells (effector-to-target ratio, 100:1), 100 μg/mL of EpMab-37-mG_2a-f or control mouse IgG_2a in 96-well plates. After incubation for 4.5 h at 37 °C, the Calcein release into the medium was measured with an excitation wavelength (485 nm) and an emission wavelength (538 nm) using a microplate reader (Power Scan HT; BioTek Instruments, Inc., Winooski, VT, USA).
Cytolyticity (% lysis) was determined as follows: % lysis = (E − S)/(M − S) × 100. “E” is the fluorescence in the presence of both effector and target cells. “S” is the spontaneous fluorescence in the presence of only target cells. “M” is the maximum fluorescence by the treatment with a lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM of EDTA, and 0.5% Triton X-100).

The hydrogen peroxide-scavenging ability of Ser-PAMAM-Cys was evaluated by the BES-H₂O₂ probe method with a slight modification [16 (link)]. In brief, Ser-PAMAM-Cys and Cys were added to 1,000 µM hydrogen peroxide at concentration of 250 µM (as the thiol concentrations in PBS) and incubated at 37 °C for 1 h in the dark. About 20 µL each of the mixture and 5 µM BES-H₂O₂ were reacted for 30 min. The fluorescence intensity of these samples was measured in a microplate reader (PowerScan HT, BioTek Instruments, Inc., Winooski, VT, USA) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

Zymomonas mobilis TISTR 548 cells were grown on YPD medium at 39°C. At 12 h, 5 μM H₂DCFDA was added to the first culture, and further cultivation was performed at 39°C for 30 min. Then, cells were harvested by low-speed centrifugation and washed once with phosphate-buffered saline [130 mM NaCl, 10.8 mM Na₂HPO₄, 4.2 mM NaH₂PO₄ (pH 7.2)]. The washed cells were disrupted by sonication for 30 min using an ultrasonic cell disruptor (Bioruptor; Cosmo Bio, Tokyo, Japan) and subjected to low-speed centrifugation. Supernatant fluorescence was measured using a microplate reader (POWERSCAN^® HT; BioTek Instruments, Inc., Winooski, VT, United States). Protein concentration was determined using the Lowry method (Dulley and Grieve, 1975 (link)). The result obtained for intracellular reactive oxygen species (ROS) levels is expressed as fluorescence intensity per protein concentration, and the ratio of the number of cells grown in the presence of a metal ion to that of cells grown in its absence was estimated and expressed as percentage.

ADCC assay was performed as previously described [16 (link),19 (link),20 (link),21 (link),22 (link),23 (link),24 (link),25 (link),26 (link)]. Briefly, canine mononuclear cells derived from dog blood were obtained from Yamaguchi University and resuspended in DMEM with 10% FBS to be used as effector cells. Target cells (D-17 and A-72) were labeled with 10 μg/mL calcein AM (Thermo Fisher Scientific, Inc.) and resuspended in the same medium. The target cells (2 × 10⁴ cells/well) were plated in 96-well plates and mixed with effector cells (effector/target cells ratio, 50:1), 100 μg/mL of E134Bf or the control dog IgG. After 4.5 h of incubation at 37 °C, the release of calcein into the supernatant was measured in each well. The fluorescence intensity was determined using a microplate reader (PowerScan HT; BioTek Instruments, Inc., Winooski, VT, USA) with excitation and emission wavelengths of 485 and 538 nm. Cytolytic activity (% lysis) was calculated as follows: % lysis = (E − S)/(M − S) × 100, where E denotes the fluorescence measured of the combined cultures of the target and effector cells; S, the spontaneous fluorescence of the target cells only; and M, the maximum fluorescence measured following the lysis of all cells with a buffer containing 0.5% Triton X-100, 10 mM Tris-HCl (pH 7.4), and 10 mM EDTA. All experiments were conducted in triplicate.

Intracellular calcium concentrations were measured by detecting the fluorescence of cells treated with a calcium-sensitive indicator, Fluo-4 AM [54 (link)]. L6 cells harvested 10 d after differentiation were replated in a 96-well plate (Iwaki, Tokyo, Japan) at 1.5 × 104 cells/well for 24 h. Subsequently, the Ca²⁺ levels were determined using a Calcium Kit II-Fluo 4 (Dojindo, Kumamoto, Japan) using Powerscan HT (BioTek, VT, USA). Briefly, cells were washed twice with non-serum medium containing 2.5 mM probenecid 24 h after replating. The cells were incubated with 4 μg/mL Fluo-4 AM and 0.025% (w/v) pluronic F-127 for 30 min in the dark at 37 °C. After washing twice with non-serum medium, cells were measured using a Powerscan HT instrument with an excitation band of 485/20 nm, and fluorescence intensity was measured at 528/20 nm. Baseline signals (F0) were recorded 5 min before the addition of each stimulus. Continuous fluorescence measurements were performed for 20 min. The results are shown as F/F0 ratios after background subtraction, where F is the fluorescence signal intensity and F0 is the baseline intensity, as calculated from the average of five frames before stimulus application [54 (link)].

Cell proliferation in vitro was measured using MTS tetrazolium (Cell Titer 96 Aqueous, Promega, Madison, WI, USA). Cells were plated (500 cells/100 μL/well) in triplicate in 96-well plates. Cell viability was measured every 24 h for 96 h. After adding 20 μL of MTS to the wells followed by a 1-h incubation at 37°C, the absorbance at 490 nm (reference, 630 nm) was read using a microplate reader (Power Scan HT, Bio Tek Instruments, Winooski, VT, USA). The mean absorbance of the 3-well set was obtained from 0 to 96 h. Statistical significance was analyzed using the standard Student's t-test. P-values <0.05 were considered statistically significant.