Shear flow assays were performed as previously described [4 (link), 20 (link)]. 6-channel μ-slides (Ibidi) were coated with protein A and VCAM-1/Fc and co-immobilized with/without CXCL12 at 4°C overnight, washed, and blocked with 2% human serum albumin. Isolated CD49d+ CLL cells were incubated with/without AMD3100 (5 μM) or anti-CD49d mAb (1 μg/ml) for 10 min. The cells were perfused in the flow chamber, allowed to accumulate at sub-physiological shear stress (0.5 dyn/cm2) to the coated substrates, and then subjected to physiological shear stress (2 dyn/cm2). The entire perfusion period was recorded and digitalized. Analysis of the video-recorded segments was done using a custom designed image analysis software (Wimasis). Frequencies of adhesive categories were determined as percentages of cells flowing directly over the substrates. All experiments were performed at 37°C.
6 channel μ slide
Manufactured by Ibidi
Sourced in Germany
The 6-channel μ-Slides are a specialized laboratory equipment designed for cell-based assays. They feature six independent channels, allowing for the simultaneous cultivation and observation of multiple cell samples. The μ-Slides are constructed with high-quality materials and are suitable for various microscopy techniques.
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Lab products found in correlation
Ixplore spinsr, by Olympus (4 mentions)
Corrsight microscopy, by Thermo Fisher Scientific (3 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Fluo 8 am, by Abcam (2 mentions)
Observer d1 microscope, by Zeiss (1 mentions)
Fugene hd, by Roche (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Ba575if filter, by Olympus (1 mentions)
Confocal imaging system, by Olympus (1 mentions)
Ab113851, by Abcam (1 mentions)
Cellsens acquisition software, by Olympus (1 mentions)
Oxidative stress detection reagent, by Abcam (1 mentions)
Superoxide detection reagent, by Abcam (1 mentions)
Cellular ros superoxide detection assay kit, by Abcam (1 mentions)
Las af software, by Leica camera (1 mentions)
Hoechst 33342 dye, by Thermo Fisher Scientific (1 mentions)
Anti irf1, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 conjugated goat anti mouse igg h l secondary antibody, by Thermo Fisher Scientific (1 mentions)
Tcs sp5 confocal microscope, by Leica camera (1 mentions)
Oil immersion objective, by Olympus (1 mentions)
15 protocols using 6 channel μ slide
1
Shear Flow Assay for CLL Cell Adhesion
2
Fluorescence Microscopy of Transfected Cells
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Cells were cultured on coverslips or in 6-channel μ-slides (Ibidi) for one day. Plasmids were transfected using Fugene HD (Roche) as described in Figure Legends. After 24 h, cells remained untreated or were treated as indicated in the manuscript text and Figure Legends. Cells were fixed with 3.7% paraformaldehyde in 1xPBS (pH 7.4) and permeabilized with 0.1% saponin or with 0.3% Triton X-100 in 1xPBS. GFP-tagged constructs (green) were directly detected by fluorescence microscopy, using LipidTox (red) and DAPI (4′,6-diamidino-2-phenylindole, blue) to visualize LDs and cell nuclei, respectively. Flag-tagged constructs were detected by immunocytochemistry with anti-flag antibody, followed by Alexa Fluor 647-conjugated secondary antibody. Images were taken with a Zeiss Observer.D1 microscope or with a Zeiss LSM 510 META confocal microscope.
3
Analyzing Neutrophil-Endothelial Interactions
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Use of human umbilical vein endothelial cells (HUVEC) was approved by the East London & The City Local Research Ethics Committee (REC Ref 06/Q0605/40 ELCHA, London, U.K.). HUVEC were cultured onto 6-channel μ-slides (IBIDI, Germany) coated with gelatin until confluence and stimulated with 10 ng/ml tumour necrosis factor-α for 4 (Sigma–Aldrich) [10 (link)]. Patient neutrophils were isolated via density gradient as previously described [10 (link)]. Isolated cells were incubated with vehicle or 10 nM human recombinant AnxA1 for 10 min at 37°C, before flow over HUVEC monolayers at a shear stress of 1 dyne/cm2, for 8 min, as previously described [10 (link)]. Neutrophil/HUVEC interaction in the flow chamber was monitored on six random fields recorded for ten seconds. Analysis of total cell capture, rolling and firmly adherent neutrophils was carried out off-line by manual quantification using Image-Pro Plus software (Media Cybernetics, Inc. Bethesda, MD U.S.A.).
4
Confocal FRET Imaging of 6HB Proteins
5
Quantifying Cellular ROS Levels
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The level of ROS was measured using DCFDA dye (ab113851, Abcam) according to the manufacturer’s instructions. In brief, SH-SY5Y cells were seeded on 6-channel μ-Slides (80606, Ibidi) and differentiated for 5 days. After treatment, culture medium was removed and cells were loaded with 20 µM DCFDA diluted in dilution buffer for 30 min at 37 °C. Cells were then washed 3 times with culture medium and imaged under FEI CorrSight microscopy. Fluorescence intensity was quantified with ImageJ.
6
Immunofluorescence Staining of Cathepsin B and LAMP1
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Immunofluorescence staining was performed to assess the colocalization of cathepsin B with the lysosomal marker LAMP1. Cells were seeded in 6-channel μ-Slides (Ibidi, Gräfelfing, Germany) at a density of 10 000 cells per well and co-treated with either oleic or palmitic acid and IONPs according to the scheme presented in Fig. 1b . Subsequently, the cells were washed with PBS and fixed with 4% paraformaldehyde in PBS pH 7.4 at room temperature for 10 min. Then the cells were permeabilized with 0.5% Triton X-100. Immunofluorescence staining was performed on fixed cells using primary antibodies against cathepsin B and LAMP1 and AlexaFluor 568- or AlexaFlour 488-conjugated secondary antibodies. The dilutions and catalogue numbers of the primary and secondary antibodies used are provided in ESI Table S2.† The stained cells were imaged using spinning disk confocal microscopy IXplore SpinSR (Olympus, Tokyo, Japan). ImageJ software (NIH, Bethesda, MD, USA) was used for image processing and quantification.
7
Mitochondrial Membrane Potential Assessment
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Cells were seeded in 6-channel μ-Slides (Ibidi, Gräfelfing, Germany) at a density of 10 000 cells per well and co-treated with either oleic or palmitic acid and IONPs according to the scheme presented in Fig. 1b . Subsequently, the cells were stained with 1 μM JC-1 probe and imaged using spinning disk confocal microscopy IXplore SpinSR (Olympus, Tokyo, Japan). A treatment with 20% ethanol for 30 minutes was used as a positive control. JC-1 is a highly selective and sensitive dye to assess mitochondria potential. It enters the mitochondria and undergoes a reversible color change from red to green as the membrane potential decreases.64 (link) In cells with high mitochondrial membrane potential (ΔmΦ), JC-1 forms complex J-aggregates that possess intense red fluorescence. In cells with low ΔmΦ, JC-1 remains in the monomeric form, which displays mostly green fluorescence. Therefore, the ratio of green to red fluorescence is used as an indicator of ΔmΦ changes. This ratio is not affected by other factors such as mitochondrial size, shape, and density, which may influence single-component fluorescence signals.64 (link)
8
Visualizing Lipid Droplet-Lysosome Interactions
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To analyze the interactions between lipid droplets and lysosomes in living cells, we utilized an IXplore SpinSR Olympus super-resolution imaging system (Olympus, Tokyo, Japan). Cells were seeded in 6-channel μ-Slides (Ibidi, Gräfelfing, Germany) and incubated with palmitic acid and IONPs according to the scheme presented in Fig. 1b . Then the cells were stained for lipid droplets (LipidSpot™ 488 Lipid Droplet Stain) and lysosomes (LysoTracker™ Red DND-99). The catalog numbers of the fluorescent probes used are provided in ESI Table S2.† Fluorescence images were acquired using cellSens acquisition software (Olympus, Tokyo, Japan). ImageJ software (NIH, Bethesda, MD, USA) was used for image processing and analysis of the gray value intensities of lysosomes and lipid droplets.
9
Quantifying Cellular ROS and Superoxide Levels
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For the detection of reactive oxygen species (ROS) levels, we used a Cellular ROS/Superoxide Detection Assay Kit (Abcam, Cambridge, United Kingdom). Briefly, cells were seeded in 6-channel μ-Slides (Ibidi, Gräfelfing, Germany) at a density of 10 000 cells per well and co-treated with either oleic or palmitic acid and IONPs according to the scheme presented in Fig. 1b . Afterwards, the cells were stained with the Oxidative Stress Detection Reagent (green) for ROS detection and the Superoxide Detection Reagent (orange) according to the manufacturer's instructions (Abcam, Cambridge, United Kingdom). The stained cells were imaged using spinning disk confocal microscopy IXplore SpinSR (Olympus, Tokyo, Japan). As a positive control treatment with 1 mM H2O2 for 30 min was used. For quantitative analysis, the corrected total cell fluorescence (CTCF) of the full area of interest (ROI), i.e., selected region of a single cell, was calculated. The CTFC intensity of the single cell for separate “green” and “red” fluorescence channels was calculated for each image utilizing a previously described method.56 (link) CTCF = integrated density − (area of selected cell × mean fluorescence of background readings). For background readings, we utilized a region placed in an area without fluorescent objects. Image quantification was performed using ImageJ software (NIH, Bethesda, MD, USA).
10
Live Cell Imaging for Mitochondrial Dynamics
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For live cell imaging, we used FEI CorrSight microscopy equipped with wide-field and an Andromeda spinning disk. The FEI MAPS software, in conjunction with Live Acquisition software (LA, FEI) was used to control the microscope and to capture still and time-lapsed images. The microscope was further equipped with an incubation system able to control temperature (Ibidi) and CO2 (Digital Pixel, UK) levels. Cells were seeded in 6-channel μ-Slides (Ibidi, 80606). To monitor the cells over time, cell death stimuli were introduced to the cells through perfusion tubes (Ibidi), which were connected to the cell chamber. When cell death stimuli were removed, fresh medium was then introduced to the chamber through these tubes. Fluorescence signals of nuclei, mitochondria, and Cyto.c-GFP were visualized by Andromeda spinning disk. Z-stacks consisted of 10 planes with a Z-interval of 1 μm. Images were shown in the merged z-stacks of 10 planes with the maximum fluorescence intensity. Mock-treated cells were imaged in parallel to ensure that imaging and staining procedures were not cytotoxic. We define mitochondrial fragmentation as the mitochondria in cells appearing as small globes, lacking a tubular and interconnected network as previously reported51 (link). Representative images were edited in ImageJ software.
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