Biotek synergy 4

Cells were seeded in triplicate in 12-well plates 24 h prior to treatment, pretreated with SPIOs@PLGA@Au or antiPD-L1-SPIOs@PLGA@Au for 24 h, and then irradiated. After irradiation, fresh medium containing 4 μM CM-H2DCFDA (ThermoFisher, C6827) for ROS measurement was added to each well. After incubation for 30 min in a humidified incubator (at 37 °C, 5% CO2), the cells were washed with PBS and trypsinized to obtain a cell suspension. ROS was analyzed by Bio-Rad microplate reader (Biotek Synergy 4) at 488 nm or visualized by CLSM.

Each antigen was screened for sero-reactivity to each of 36 plasma (‘antisera’) collected at each of the three timepoints (A, C1 and C2). Thirty-two of these plasma samples came from the same children from which the 36 recombinant proteins derived. Sero-reactivity was measured by coating 96-well Nunc^TM Maxisorb^TM immunoplates plates overnight at 4°C with recombinant protein diluted in Tris-buffered saline (TBS) at a concentration of 1μg/ml. Plates were blocked with 3% bovine serum albumin (BSA) in TBS, washed and then incubated in duplicate at a 1:200 dilution of individual sera diluted in 1% non-fat milk powder in TBS. After incubation for 2h at room temperature, plates were washed, then bound antibody was detected with mouse anti-human IgG alkaline phosphatase antibody or AffiniPure donkey anti-human IgM alkaline phosphatase antibody (Jackson ImmunoResearch Laboratories, Inc.) diluted 1:5000 in 1% non-fat milk powder in TBS. The reaction was developed using o-Phenylenediamine dihydrochloride (Sigma-Aldrich) and then stopped with 2M sulphuric acid. Optical density (OD) was read at 450nm in an ELISA microplate reader (BioTek Synergy 4, BioTek Instruments).

Spectrophotometry was the method used to evaluate the total phenolic content (TPC) and condensed tannin content (CTC). TPC was determined by the Folin-Ciocalteu assay [66 (link)], with modifications. Absorbance was measured at 725 nm and gallic acid was used as a standard. Results were expressed in gallic acid equivalents per gram of dried extract (mg GAE g⁻¹ dw). Tannin content (CTC) was evaluated by the colorimetric method of 4-dimethylaminocinnamaldehyde hydrochloric acid (DMACA-HCl) [67 (link)], with adaptations created by Zou et al. [68 (link)] for 96-well microplates. Absorbance was measured at 640 nm and the results were expressed in milligrams of catechin equivalents per gram of dried extract (mg CE g⁻¹ dw). All assays were performed in a microplate reader (Biotek Synergy 4, Biotek Instruments, Winooski, VT, USA).

Experiments were performed
in 384-well black polypropylene PCR plates (Axygen) in 10 μL
volume. In each well, 9 μL of compound solutions in buffer (20
mM HEPES (pH 7.4), 150 mM NaCl, 1 mM TCEP, 0.005% Tween-20 (v/v),
1% DMSO (v/v)) were diluted, followed by the addition of 1 μL
of 30 μM HDAC6^1109–1213 or 10 μM HDAC6^1–1215 and 500 nM N-terminally FITC-labeled RLRGG or
1 μL of 2 μM HDAC6^1109–1213 and 500
nM N-terminally FITC-labeled LRLRGG were added to each well. The LRLRGG
substrate was used for compounds with K_D <∼1 μM. Following 1 min centrifugation at 1000 RPM,
the plate was incubated for 10 min before FP measurements with a BioTek
Synergy 4 (BioTek) at excitation and emission wavelengths of 485 and
528 nm, respectively. The data was processed in GraphPad Prism using
Sigmoidal, 4PL, X is log(concentration) fit.

The final concentration
of 200 nM HDAC6^1–1215 was incubated with compound
dilutions prepared with 20 mM HEPES (pH 8.0), 1 mM MgCl₂, 137 mM NaCl, 2.7 mM KCl, 0.05% BSA (v/v), and 2% DMSO (v/v) in
a 384-well microplate (Greiner). A final concentration of 50 μM
Boc-Lys(TFA)-AMC was added and incubated for 1 hour at room temperature,
followed by the addition of developer solution containing a final
concentration of 50 μM TSA. Following a 1 min centrifugation
at 1000 RPM, fluorescence intensity was read using BioTek Synergy
4 (BioTek) with excitation and emission of 360 and 460 nm. The data
were analyzed with GraphPad Prism 8.2.0.