Hoechst

Intracellular ROS production was detected using the ROS Assay Kit (Beyotime, S0033). Briefly, cells were initially seeded in a 96-well plate at a density of 1 × 104 cells/well and subsequently treated with or without 10 μM Aβ. After completion of the treatment, the cells were co-stained with 10 μM DCFH-DA and 3 μg/mL Hoechst (Beyotime, C1022) at 37 °C for 20 min. Then, cells were washed twice with PBS, and the ROS levels were determined using BioTek SynergyNEO (BioTek, Winusky, VT, USA) at excitation/emission wavelengths of 488/525 nm for DCFH-DA and 350/461 nm for Hoechst. Alternatively, the cells in the 96-well black plate were observed using a laser-scanning confocal microscope (Operetta, Perkin Eimer, Waltham, MA, USA).

HepG2 cells were seeded in 96-well plates at 4 × 10³ cells/well and adhered overnight. The medium was changed to DMEM supplemented with 62 µM sodium oleate (Sigma-Aldrich, St. Louis, MO, USA) to induce the presence of lipid droplets [42 (link)] and two different concentrations of plant extracts (final concentration of 30, 10 µg/mL; dilution 1:200 of stock concentration). After 6 h, cells were stained with 75 ng/mL Nile red (Sigma-Aldrich) and 10 µg/mL Hoechst 33342 (Sigma Aldrich) in HBSS. After incubating at 37 °C for 10 min and in the absence of light, cells were washed twice with HBSS. Fluorescence was read in a Synergy HT multi-detection microplate reader (Biotek) at 485/572 nm excitation/emission for Nile red and 360/460 nm for Hoechst. Following this, cells were fixed with ice-cold trichloroacetic acid (TCA) for 1 h at 4 °C in the dark and then washed four times with ddH₂O to remove all the TCA. After being air-dried, the cells were stained with sulforhodamine B (SRB) in 1% acetic acid for 15 min and rinsed quickly with 1% acetic acid five times to remove unbound SRB. Plates were air-dried and bound dyes were solubilized with 150 µL Tris–HCl (10 mmol/L, pH 10.5) before reading the absorbance at 492 nm with the reference at 650 nm in a multi-detection microplate reader (Biotek, Synergy HT).

Animals were euthanized under anesthesia by exsanguination via cardiac puncture, the right kidney was removed, fixed in 10% formalin (vol/vol) and processed for histology. Hematoxylin and eosin (H&E) staining was performed on 4 μm kidney paraffin sections.
The expression of human C-peptide and glucagon in the implants were assessed by immunofluorescence using specific antibodies. Sections were dewaxed and hydrated, boiled in citrate buffer (1X) at 100 °C for 20 min and then incubated with PBS-0.1% Tween-0.1% bovine serum albumin (wt/vol) for 30 min at room temperature to block the unspecific reactivity. Subsequently, sections were incubated overnight with the specific antibodies: anti-human C-peptide and anti-human glucagon (1:200; Abcam). In the negative control the primary antibodies were omitted or substituted with non-immune human IgG. Then the sections were incubated with the corresponding secondary antibodies (1:500 Goat anti Rabbit Alexa Fluor 488 and Goat anti mouse Texas Red (Invitrogen), subsequently with Hoechst 1:500 (Dako) for 10 min and then mounted with Fluoromount (Sigma). Microscopy analysis was done using a Cell Observer SD-ApoTome laser scanning systems with 20X objective (Carl Zeiss). Analysis was performed in a blinded fashion with AxioVision 4.8 software (Carl Zeiss).

A XF96 extracellular flux analyzer (Agilent Technologies) was applied to determine the bioenergetic profile of intact cells. Hematopoietic stem cells were harvested, seeded onto poly-d-lysine–coated XF96 plates (7 × 10⁴ cells per well), and incubated in RPMI medium (Agilent Technologies; supplemented with 5 mM glucose, 2 mM l-glutamine, and 1% FBS) for 1 hour in a CO₂-free incubator. Oxygen consumption rate (OCR) was first measured in basal conditions (named as basal respiration) before cells were sequentially treated with 1.5 μM oligomycin (MilliporeSigma), 1 μM FCCP (MilliporeSigma), and 0.5 μM rotenone/antimycin A (MilliporeSigma) and 8 μM Hoechst (Thermo Fisher Scientific). Recorded OCR after all treatments was defined as non-mitochondrial respiration, which was subtracted from all OCR parameters. ATP production was calculated by the difference between the basal and oligomycin-inhibited respiration; maximal respiratory capacity was obtained as the rate of respiration after uncoupler FCCP treatment. Hoechst-positive cells were counted by a Cytation machine (Agilent). The number of live cells was normalized to individual sample for a final evaluation of sample OCR by Wave software (version 2.6.3, Agilent).