Hcs cellmask deep red stain

NB69 (100.000 cells/well) and CHP134 (150.000 cells/well) were seeded in a six-well plate and treated with different concentrations of Cou6-NPs (NB69: NT, 0.0003 µg/µL, 0.003 µg/µL, and 0.03 µg/µL; CHP134: NT, 0.003 µg/µL) for 48 h. After a wash with PBS, cells were fixed with a paraformaldehyde solution (4% v/v final, 15 min incubation at room temperature), followed by two washes with PBS. Hoechst 33342 (1 μg/mL, Thermo Fisher Scientific) and HCS CellMask™ Deep Red Stain (Thermo Fisher Scientific, H32721, 1:2000, 20 min at room temperature) were used to identify cell nuclei and cell surfaces, respectively. The fluorescence signal in NB69 cells was detected using the Operetta High Content Imaging System (PerkinElmer) and quantified using the Harmony software 4.1 (PerkinElmer). The CHP134 cells were imaged with a Leica TCS SP8 confocal microscope equipped with a 63×/1.4 oil objective and the proper laser/filter setting. Images were acquired at 400 Hz unidirectional scan speed with 2× zoom and a 130 nm z-step.

A PROTEOSTAT Aggresome Detection kit (Enzo Life Sciences Inc.) was used to detect aggresomes in cells, in accordance with the manufacturer’s instructions. Nuclei were counterstained with Hoechst. Cytoplasm was stained with HCS CellMask Deep Red Stain (Thermo Fisher Scientific Inc.). All images were collected with an IN Cell Analyzer 6000. To assess the effects of different chemical compounds on aggregate accumulation, cells were cultured with each compound and aggregate intensities were evaluated. The compounds used in these analyses included trimethylamine N-oxide (TMAO, Sigma-Aldrich), sodium 4-PBA, Sigma-Aldrich), geldanamycin (TCI, Tokyo, Japan), 2-hydroxypropyl-β-cyclodextrin (Sigma-Aldrich), rapamycin (Sigma-Aldrich), and valproic Acid (VPA, FUJIFILM Wako Pure Chemical Corporation).

Cells were seeded at 12,500 cells/well/50 µL in a 384-well plate (CellCarrier Ultra), in the presence of 25 ng/mL PMA. After 24 h, cells were treated with hemin, 3,4DHPAA, or 4HPAA with or without 5 pg/mL LPS and 10 ng/mL INFγ (Scheme 2). After 72 h, cells were fixed in 4% w/v PFA solution for 15 min and washed 3× with PBS. Cells were blocked for 2 h, then incubated overnight at 4 °C with anti-HIF-1α (1/200, rabbit pAb, Cat# NB100-134, Novus Biologicals, Littleton, CO, USA) prepared in PBS. After washing 3× with PBS, cells were incubated for 2 h at room temperature with 10 μM Hoechst 33342, 0.2 μg/mL HCS CellMask™ Deep Red Stain, and Alexa Fluor 488 goat anti-rabbit IgG (H+L) (1/400, Cat# A11008, ThermoFisher), all prepared in PBS. Cells were washed 3× in PBS and imaged on the Opera Phenix™ High Content Screening System using a 20× water objective, in which nine fields were imaged per well. Images were analyzed using Harmony software and fluorescence of Alexa Fluor 488 was quantified in the nucleus and cytosol areas [50 (link)].

Immunofluorescence staining of formaldehyde-fixed cells (2%, 20 min) in 96-well plates was conducted as described previously [57 (link)]. A rabbit-anti-Slc1 serum (1:400, described elsewhere [58 (link)]) in combination with AlexaFluor555- or AlexaFluor647-labeled secondary antibodies (Thermo-Fisher-Scientific) was used for the detection of C. trachomatis. When indicated, DNA was stained for 10 min with 2–10 µg/ml Hoechst 33342 (Thermo-Fisher-Scientific) and cells were stained for 10 min with either 5 µM CellTrace CFSE (Thermo-Fisher-Scientific) or 2 μg/ml HCS CellMask Deep Red stain (Thermo-Fisher-Scientific). Images were acquired on an ImageXpress Micro XL system (Molecular Devices) or on a Cellomics ArrayScan VTI HCS imaging system (Thermo-Fisher-Scientific). Automated image analysis (such as for the determination of the percentage of infected cells, inclusion numbers, or average fluorescence intensities per cell) was conducted using MetaXpress 5.3.0.4 (Molecular Devices) or HCS Studio Cell Analysis Software (Thermo-Fisher-Scientific), respectively.

HCT116 cells were seeded in 384-well plates then transfected with mRNA encoding bioPROTACs. Following 18 h incubation, cells were washed in PBS, fixed for 15 min at room temperature in 4% methanol-free formaldehyde (ThermoFIsher Scientific) then blocked in 3% BSA and 0.1% Triton X-100 for 1 h. Cells were stained overnight with mouse anti-HuR (ThermoFisher Scientific) and rabbit anti-HA (Abcam) antibodies. Nuclei were stained with Hoechst (ThermoFisher Scientific) and the entire cell was stained with HCS CellMask™ Deep Red Stain (ThermoFisher Scientific). Cells were visualised using the CV7000 spinning disk confocal microscope (Yokogawa Inc.) using a 20× objective and 2 × 2 binning. Analyses were undertaken using Columbus software (PerkinElmer) to quantify HuR abundance. Due to some evidence of epitope competition between the VHH^HuR and HuR antibody, immunofluorescence was only used as an orthogonal approach.

Immunocytochemistry was performed as described previously, with some modifications [20 (link)]. Mitochondria were stained by exposing cells to MitoTracker Red CMXRos (100 nM, Thermo Fisher Scientific Inc.) for 15 minutes before fixation with 4% paraformaldehyde/phosphate-buffered saline (PBS). Cells were then permeabilized with 0.2% Tween 20/PBS for 15 minutes. Next, cells were blocked with 5% fetal bovine serum (FBS)/PBS for 30 minutes. Cells were then incubated at 4°C for 16 hours with rabbit anti-TOM20 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA) primary antibody. After washing with PBS, cells were incubated for 60 minutes with goat anti-rabbit secondary antibody conjugated with Alexa Fluor 488 (1:200; Thermo Fisher Scientific Inc.). Nuclei were counterstained with Hoechst (Dojindo). Cytoplasm was stained with HCS CellMask Deep Red Stain (Thermo Fisher Scientific Inc.). All images were collected with an IN Cell Analyzer 6000.