96 well pcr plate

Mice were sacrificed at the indicated ages by cervical dislocation. Ovaries were extracted and immediately placed in cryo-buffer containing 50% PBS, 25% ethylene glycol and 25% DMSO (Sigma–Aldrich, Austria) and stored at −80°C. For oocyte extraction, ovaries were placed into a drop of cryo-buffer and disrupted using scalpel and forceps. Oocytes were collected and remaining cumulus cells were removed mechanically by repeated careful suction through glass capillaries. Prepared oocytes were then washed in PBS before they were individually placed into compartments of 96-well PCR plates (Life Technologies, Austria) containing 10 μl of oocyte-lysis buffer (Lee et al., 2012 (link)) (50 mM Tris-HCl, [p.H 8.5], 1 mM EDTA, 0.5% tween-20 [Sigma–Aldrich, Austria] and 200 μg/ml Proteinase K [Macherey–Nagel, Germany]). Samples covered stages from primary oocytes of 3 day-old mice up to mature oocytes of 40 day-old mice. Samples were lysed at 55°C for 2 hr, and incubated at 95°C for 10 min to inactivate Proteinase K. The cellular DNA extract was finally diluted in 190 μl Tris-EDTA buffer, pH 8.0 (Sigma–Aldrich, Austria). 3 μl were used per qPCR reaction.

NP_105–113-B*07:02-specific CD8⁺ T cells were stained with phycoerythrin (PE)-conjugated HLA-B7 NP_105–113 pentamer (ProImmune). Live/dead fixable Aqua dye (Invitrogen) was used to exclude nonviable cells from the analysis. Cells were washed and stained with the following surface antibodies: CD3-FITC (BD Biosciences), CD8-PercP-Cy5.5, CD14-BV510, CD19-BV510 and CD16-BV510 (BioLegend). After exclusion of nonviable/CD19⁺/CD14⁺/CD16⁺ cells, CD3⁺CD8⁺pentamer⁺ cells were sorted directly into 96-well PCR plates (Thermo Fisher Scientific) using a BD Fusion sorter or BD FACS Aria III (BD Biosciences) and stored at −80 °C for subsequent analysis.

The binding affinity of the synthesized compounds to M. smegmatis MshC (Ms-MshC) was evaluated by applying a fluorescence-based thermal shift assay (TSA). A 20 μL reaction system containing 0.2 mg/mL protein, 1× thermal shift dye (ThermoFisher Scientific, Waltham, MA, USA), 50 mM HEPES pH 7.0, 150 mM KCl, 10% (v/v) ethylene glycol and various concentrations of each compound was prepared in 96-well PCR plates (ThermoFisher Scientific, Waltham, MA, USA) on ice. The plates were centrifuged to remove air bubbles and then measured by using the Applied Biosystems real-time PCR system (ThermoFisher Scientific, Waltham, MA, USA) with an excitation filter of 580 ± 10 nm and an emission filter of 623 ± 14 nm. The plates were gradually heated from 4 °C to 95 °C at a rate of 0.05 °C/s. The melting curves were fitted with a Boltzmann model using the Protein Thermal Shift software to calculate the protein melting temperature (T_m). Due to the compounds containing a cysteine group, which is easily oxidized, forming a disulfide, the reducing agent TCEP (1 mM) was added to eliminate the effects of oxidation. The compared results are shown in Table 1. Triplicate assays were applied to controls, and all compounds and the averaged T_m were used.

The q-ORO assay was done as described [104 (link)]. Age-synchronized Bristol N2 wild type nematodes in the L4-larval stage were exposed for 24 h to toxic solutions of BPA, PPB, and TCS. To prepare the dye solution, a 0.5% oil red O (MP, Cat. No. 155984) stock solution was prepared in high-quality 100% isopropanol, incubated at room temperature for a day, and then filtered through a 0.45 µm filter. The stock was freshly diluted to 60% with filtered water the day before its use, and it was then incubated at room temperature overnight. The stock was filtered through a 0.45 µm filter. Two hundred microliters of high-quality 60% isopropanol was added to the worms in the 96-well PCR plates (Thermo Scientific, Waltham, MA, USA). Worms were settled to the bottom of the wells, and then they were aspirated with up to 175 µL of buffer. Two hundred microliters of freshly filtered ORO working solution was added, which sealed the plates. Worms were stained for 6–18 h at 25 °C; after 6–18 h, the supernatant was aspirated. Then, 100 µL of 0.01% Triton X-100 was added in the S buffer. Images were recorded by using an optic microscope [104 (link)].

The apparent melting temperature (T_m) of the TaTT variants was determined using the Thermofluor (also known as a Differential Scanning Fluorimetry (DSF)) assay. The samples diluted to 0.5 mg/ml in 50 mM sodium phosphate buffer, pH 8.0, containing 200 mM NaCl, 20 μM PLP and 1000× dilution of Sypro Orange dye (Sigma-Aldrich), were transferred to a 96-well PCR plates (Thermo Fischer Scientific) in the final volume 50 μl per well. Buffer mixed with Sypro dye was used as a negative control. Plates were placed in a real-time cycler (Bio-Rad C1000, with the CFX96 Real-Time accessory, USA), and the temperature was ramped up from 20 to 95°C in 1.0°C min^-1. Relative fluorescence was measured using the FRET channel, and GraphPad Prism v8.0 (GraphPad Software Inc., USA) was used to fit the collected data a sigmoidal curve and calculate T_m using Boltzmann model. Assays were run in triplicate, and T_m was reported as the average of the three runs.

Whole postmortem brain specimens were processed as previously described^{32 (link),44 (link)}. For RNA-sequencing experiments, frontoinsula (FI) was identified on frozen cortex slabs of interest, and the region of interest was removed and vibratome sectioned at 500 µm intervals^{32 (link),44 (link)} (Fig. 1). Layer 5 was microdissected from vibratome sections stained with fluorescent Nissl and nuclei were isolated from microdissected tissue pieces using Dounce homogenization. Mouse monoclonal anti-NeuN antibody (EMD Millipore, MAB377) was applied to nuclei preparations followed by secondary antibody staining (goat anti-mouse Alexa Fluor 594, ThermoFisher), and single-nucleus sorting was carried out on a BD FACSAria Fusion instrument (BD Biosciences) using a 130 µm nozzle following a standard gating procedure (Supplementary Fig. 1)^{32 (link),44 (link)}. Approximately 10% of nuclei were intentionally sorted as NeuN–negative to capture non-neuronal cell types. Single nuclei were sorted into 96-well PCR plates (ThermoFisher Scientific) containing 2 µL of lysis buffer (0.2% Triton-X 100, 0.2% NP-40 (Sigma Aldrich), 1 U/µL RNaseOUT (ThermoFisher Scientific), PCR-grade water (Ambion), and ERCC spike-in synthetic RNAs (Ambion). 96-well plates were snap frozen and stored at −80 °C until use. Positive controls were pools of 10 nuclei, 10 pg total RNA, and 1 pg total RNA.