Operetta cls system

Hepatic stellate cells (LX2) or Endothelial cells (HUVECs) were seeded at 2.5 × 10³ cells/well in 384-well microplate and incubation for 16 h at 37 °C in a humidified incubator of 5% CO₂. LX2 and HUVECs were treated with 20 ng/ml recombinant human TGF-β1 (100-21C, Peprotech, Cranbury, NJ, USA) for activation, and compounds were treated for 48 h simultaneously. After incubation, cells were fixed with 4% PFA for 10 min at R.T. and incubated with primary antibodies against α-SMA (ab32575, 1:3000; Abcam) for 16 h at 4 °C. After being washed with DPBS for 3 times, the samples were incubated with secondary antibodies with fluorescence including Alexa Fluor 488 (A11008, 1:500; Invitrogen) and Alexa Fluor Phalloidin 633 (A22284, 1:100; Invitrogen) for 1 h at R.T. After washing with DPBS, the images were detected using Operetta CLS system and analyzed by Harmony software (Perkin-Elmer).

To track intracellular HIV localization, iDCs were exposed to mCherry-tagged HIV-1 (250 ng p24/ml). DCs were labeled in addition using Hoechst (nucleus), MAVS-Alexa Fluor 647, CD11c-Alexa Fluor 488, and CCR5-PerCP/Cy5-5 (BioLegend) as indicated in the figures. Confocal microscopy was performed on an Operetta CLS system (Perkin Elmer), and colocalization was analyzed using xyz stacks and 3D imaging analyses using the Harmony software (Perkin Elmer). Quantification of cell numbers and spot analyses were done using the RMS Spot Analyses program of the Harmony software (Perkin Elmer). Five to seven fields containing 350 to 500 cells were analyzed, and statistical differences were calculated using unpaired Student's t test and GraphPad Prism.

The bacterial suspension (at a concentration of 10⁹ CFU/mL) was incubated with SytoRed (20 uM, dilution 1:250) (Molecular Probes, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at room temperature. After three washes with PBS, the stained bacteria were resuspended in 1 mL of RPMI without FBS and antibiotics. THP-1 cells were seeded in 96-well plates and differentiated for 48 h with PMA. Then, THP-1-derived macrophages were incubated with different concentrations of TB (10⁶, 5 × 10⁶, 10⁷, 5 × 10⁷, 10⁸ CFU/mL) for 30 min. At the end of the incubation, the cells were washed with PBS, fixed in 4% paraformaldehyde for 5 min and washed again with PBS. After that, the cells were stained with Actin Green (Molecular Probes, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA), which binds actin with high affinity. Nuclei were stained with Hoechst (Molecular Probes, Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA), diluted 1:300 in PBS, as previously described [51 (link)], and analyzed by the Operetta CLS system (PerkinElmer, Waltham, MA, USA) in the confocal mode at 63× magnification.

Treated neutrophils were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA). After blocking with 3% bovine serum albumin (BSA), the cells were stained with a rabbit monoclonal recombinant anti-human neutrophil elastase antibody (Abcam, Cambridge, United Kingdom) at 1:250 dilution. The cells were then washed with 1x PBS and stained with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, Waltham, MA, USA) at 1:2000 dilution. The cells were again washed and stained with a mix of Hoechst 33,342 nuclear stain (Invitrogen, Waltham, MA, USA) and CellMask Deep Red plasma membrane stain (Invitrogen, Waltham, MA, USA) at 1:20,000 dilution each. Stained cells were visualized using the Operetta CLS™ system (Perkin Elmer, Waltham, MA, USA). Alexa Fluor 488-positive cells were identified under 40x water objective and 55 fields/well were analyzed using Harmony 4.9 (Perkin Elmer, Waltham, MA, USA) software.

Cell proliferation assays were performed as directed by CCK‐8 kit (Dojindo, Japan). Briefly, single cell suspensions were seeded in 96‐well plates at a density of 5000 cells/well and cultured for 0, 24, 48, and 72 h, respectively, before adding 10% of CCK‐8 solution and incubated for another 2 h. The absorbance values were measured at 450 nm by microplate reader (BioTek, USA). Scratch healing assays were performed as previously described.²⁸ Cells were cultured overnight to reach 90% confluency in a 6‐well plate before scratching with a 20‐μl plastic pipette tip. The cells that migrated into the wounded areas were imaged with the Microscope (Leica, Germany). Cell migration rate was measured as follows: (original wound width ‐ new wound width) / original wound width× 100%. For TGF‐β (MCE, USA) treatment, serum‐free DMEM supplemented with TGF‐β (15 ng/ml) was added to replace the serum‐free DMEM without TGF‐β. Tracked cell migration were performed using Operetta CLS system (PerkinElmer, USA). After overnight culture, cells were administered with TGF‐β (15 ng/ml) for 3 days and were tracked for 10 h with the Operetta CLS system. Cell distribution, migration speed, and tracks were analyzed with Harmony software.