Using Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA), U2OS cells were co-transfected with pmCherry and pCAGGS vector encoding SARS-CoV-2 Spike protein. For a negative control, U2OS cells were transfected with pmCherry only. 24-hours post-transfection, U2OS cells were trypsinized, mixed with Calu-3 cells on 96-well μ-plate (Ibidi, Germany), and incubated for 24 h in the presence (40 µM; final concentration) or absence of HNP1 or RC-101, with (10%; final concentration) or without FBS. Following the incubation, the co-cultures were counterstained with 1 μg/mL Hoechst 33342 (Invitrogen, Carlsbad, CA) and imaged using Nikon inverted microscope Eclipse Ti-E (Nikon Instruments, Melville, NY). Nikon NIS Elements AR software was used to quantify fusion indices calculated as number of nuclei per individual mCherry-positive syncytium.
96 well μ plate
Manufactured by Ibidi
Sourced in Germany
The 96-well μ-plate is a laboratory equipment designed for high-throughput cell culture applications. It features a 96-well format with a working volume of 100-200 μL per well. The plate is made of tissue culture-treated polystyrene and is suitable for a variety of cell types and assays.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
Eclipse ti e inverted microscope, by Nikon (2 mentions)
Nis elements ar software, by Nikon (2 mentions)
Geltrex, by Ibidi (1 mentions)
Opera phenix high content screening system, by PerkinElmer (1 mentions)
Ficoll paque plus, by GE Healthcare (1 mentions)
Rpmi 1640, by Lonza (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
Imagexpress micro xl system, by Molecular Devices (1 mentions)
Hoechst 33342 dye, by Thermo Fisher Scientific (1 mentions)
Celltrace cfse dye, by Thermo Fisher Scientific (1 mentions)
Metaxpress image acquisition and analysis software, by Molecular Devices (1 mentions)
Syto41, by Thermo Fisher Scientific (1 mentions)
Bodipy fl vancomycin, by Thermo Fisher Scientific (1 mentions)
Harmony 4, by PerkinElmer (1 mentions)
Harmony software, by PerkinElmer (1 mentions)
Phenix high content screening system, by PerkinElmer (1 mentions)
11 protocols using 96 well μ plate
1
SARS-CoV-2 Spike Protein Syncytia Formation
2
Angiogenesis Assay using HUVEC
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A 96-well μ-plate (IBIDI) was coated with Geltrex™ diluted in fresh M199 medium (1:1 ratio) and incubated for 10 min at 4°C and for 20 min at 37°C in a humidified atmosphere containing 5% CO2. After polymerization, HUVEC were re-suspended in complete culture medium containing GeGe3 at 20 μM and VEGF at 50 ng/ml and seeded at 10.000 cells/well. Cells were photographed with ImageXpress for 10-Hrs. The images were analyzed with ImageJ using the Angiogenesis analyzer toolset. Data were presented as the total length of the tubing segments and the network stability index, which is the ratio of total segment length and the number of isolated segments.
3
Quantifying Macrophage Phagocytic Capacity
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For each experiment, 50,000 macrophage precursors were seeded and differentiated for 1 week in separate wells of an Ibidi 96-well μ-plate (Ibidi #89626). hiPSC macrophages were treated with IFN-γ (100 ng/mL) for 72 h to increase expression of endogenous LRRK2 prior to the addition of the phagocytic materials: Alexa Fluor 488-conjugated zymosan (Life Technologies #Z23373), Alexa Fluor 488-conjugated Escherichia coli (Life Technologies #E13231), and GFP-expressing S. typhimurium (Tocris, NCTC 12023, MM11-25). Oregon Green 488-conjugated αsyn fibrils (gift from Dr Kelvin Luk, University of Pennsylvania) were prepared with endotoxin depletion according to the published methods, tested for endotoxin levels, and used at a final dilution of ≤0.01 EU/mL endotoxin (a level considered negligible) (Luk et al., 2007 (link), Luk et al., 2009 (link)).
After 2 h, cells were washed, fixed, and stained as described above. Five to 20 z-stacked confocal images per well were acquired from randomized fields using an Opera Phenix High Content Screening System (PerkinElmer) with a 63× objective. Quantification of phagosomes was carried out by the Columbus Image Data Storage and Analysis System (CambridgeSoft). Detailed methods used for the analysis are described inSupplemental Information .
After 2 h, cells were washed, fixed, and stained as described above. Five to 20 z-stacked confocal images per well were acquired from randomized fields using an Opera Phenix High Content Screening System (PerkinElmer) with a 63× objective. Quantification of phagosomes was carried out by the Columbus Image Data Storage and Analysis System (CambridgeSoft). Detailed methods used for the analysis are described in
4
Spike Protein-Mediated Cell Fusion Assay
5
Phagocytosis Imaging of Macrophages
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THP1 macrophages were seeded at a density of 7 × 10 4 cells per chamber of a four-chamber culture slide and incubated with 1 mg/ml pHrodo Red Staphylococcus aureus BioParticles conjugates (Thermo Fisher #A10010) for 120 min at 37 C according to manufacturer's instructions. After washing the cells, they were fixed with 4% PFA containing 0.25% glutaraldehyde for 15 min and mounted on cover slips. Five pictures were randomly taken for each experimental condition by confocal laser scanning microscopy. IPSdMiG were seeded at a density of 2 × 10 4 cells per well of a 96-well μ-plate (ibidi) as described above. Subsequently, iPSdMiG were incubated with 0.5 mg/ml pHrodo Red S. aureus BioParticles conjugates for 90 min at 37 C according to manufacturer's instructions. Afterward, the cells were incubated with Hoechst 33342 (5 μg/ml, Invitrogen) for 10 min at 37 C and analyzed by live cell imaging using an IN Cell Analyzer 2200 system. The intensity from the collected images was measured by Image J version 1.49. The background intensity was subtracted from the image intensity and then normalized to the WT condition.
6
Peripheral Blood Mononuclear Cell Isolation and Culture
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All protocols were approved by the local ethical committees and performed in accordance with national guidelines and regulations. Human peripheral blood was collected from healthy adult volunteers who gave written consent and with specific approval from the Texas A&M University human subjects Institutional Review Board. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood using Ficoll-Paque Plus (GE Healthcare Biosciences, Piscataway, NJ), as described previously [9 (link)]. PBMC were cultured at 37 °C in a humidified incubator with 5% (vol/vol) CO2 in either 8-well glass slides (Falcon-Corning, Tewksbury, MA or EMD-Millipore, Billerica, MA) or 96 well μ-plates (ibidi, Madison, WI) with 200 μl/well at 5 x 105 cells per ml in RPMI-1640 (Lonza, Walkersville, MD) containing 100 U/ml penicillin, 100 μg/ml streptomycin (Lonza), and 10% fetal calf serum (FCS; Seradigm, Radnor, PA) [9 (link), 11 (link), 25 (link)]. Although both human AB serum and FCS can be used for human monocyte/macrophage cultures, we used FCS as it contains low levels of pentraxins and their ligands [26 (link)–31 (link)].
7
Quantification of Membrane Ruffle Dynamics
8
Quantifying Macrophage Oxidative Stress
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For ROS measurement assays, the macrophages were seeded in 96-well plates (μ-Plate 96 well [catalog no. 89626; IBIDI]). To estimate total cell ROS, the samples were treated with CellRox green fluorescent dye (Thermo Fisher Scientific) at 5 μM for 30 min. The samples were washed three times with PBS and fixed overnight with 1% PFA. Mitochondrial ROS was measured in live, uninfected macrophages using MitoSox fluorescent dye (Thermo Fisher Scientific) at 5 μM for 30 min. The samples were imaged using confocal microscopy, and the ROS signal of each cell was quantified using NIS-Elements software. Briefly, each cell was converted to a ROI, and the MFI was measured using the ROI statistics tool.
9
Mitochondrial Morphology Imaging Protocol
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The cells were transfected with the indicated plasmids for 24 h, and then reseeded into the μ-plate 96-well (ibidi, Martinsried, Germany). Before acquisition of the images, cells were stained with Hoechst 33342 dye and CellTrace CFSE dye (Life Technologies Corporation, NY, USA), and then fixed with 4% paraformaldehyde in PBS. The images were acquired by ImageXpress Micro XL System (Molecular Devices, CA, USA). The images were analyzed by MetaXpress Image Acquisition and Analysis Software (Molecular Devices, CA, USA), and the steps of the module were illustrated in Supplementary Figure S3 . The mitochondrial signals were analyzed for the average of mitochondrial length per cell.
10
Biofilm Penetration Dynamics Investigated
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Matrix components may affect biofilm survival by preventing penetration of antibiotics. We therefore investigated vancomycin penetration in biofilms expressing PIA or Embp. Biofilms were cultivated in 96-well plates (μ-plate 96-well, IBIDI) for 48 h as described above and stained for 30 min by replacing the supernatant with PBS containing 3 μg ml−1 BodipyFL vancomycin (Thermo Fisher Scientific), 10 µM SYTO41 (Thermo Fischer Scientific) and 6 µM propidium iodide (Thermo Fischer Scientific), followed by CLSM imaging with a Plan-Apochromat 63×/1.40 oil objective and excitation at 405, 488 and 555 nm.
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