A bespoke setup was established consisting of 5% CO2/air supplied from a gas cylinder (BOC) to OB1 Microfluidic flow controller (Elveflow) to control the air pressure applied to the input of a μ‐Slide I luer 0.4 or μ‐Slide VI 0.4 channels (ibidi) containing cells and media, while the output is closed with a stopper. Particular attention was paid to leave no airspace between the stopper and the media. Cells were seeded at 22,000 cells per channel and maintained in 1% FBS complete DMEM throughout the duration of the experiments. Cyclic hydrostatic pressure at 0.1 Hz of 100 mbar or 200 mbar was applied to cells.
μ slide 1 luer
Manufactured by Ibidi
Sourced in Germany
The μ-Slide I Luer is a specialized lab equipment designed for use in microscopy applications. It features a Luer connection for convenient fluid handling. The product dimensions and technical specifications are available from the manufacturer, Ibidi.
Automatically generated - may contain errors
Lab products found in correlation
Pump system, by Ibidi (2 mentions)
μ slide 6 0.4 channels, by Ibidi (1 mentions)
Tnf α, by R&D Systems (1 mentions)
Eclipse te2000, by Nikon (1 mentions)
Nucleospin rna plus extraction kit, by Macherey-Nagel (1 mentions)
Inverted microscope model ix71, by Olympus (1 mentions)
U2os cell line, by Korean Cell Line Bank (1 mentions)
Huh 7 cell line, by Korean Cell Line Bank (1 mentions)
Lm 001 17, by Welgene (1 mentions)
Lb004 02, by Welgene (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
5 protocols using μ slide 1 luer
1
Microfluidic Cyclic Pressure Assay
2
Live-cell Microscopy of Neutrophil Adhesion
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The live-cell microscopy flow model has been described [85 (link)]. In brief, human polymorphonuclear neutrophils (PMNs) were isolated from blood by 20% dextran (1:1) sedimentation at room temperature for 40 minutes. HUVECs treated with control and CD5-2 were plated onto the μ-Slide I Luer (Ibidi, Planegg, Germany) to grow to confluence. EC monolayers were then stimulated with 5 ng/mL TNF-α (R&D system, Minneapolis, MN) for 4 hours prior to shear stress treatment. The neutrophils in HUVEC medium (106 cells/mL) were added into the ibidi pump system (Ibidi, Planegg, Germany). The dynamic adhesion of neutrophils onto control or CD5-2–treated HUVECs was recorded after 6 minutes of rolling (2 dyn/cm2 constant shear stress) using an inverted phase-contrast microscope (Nikon Eclipse TE2000, Nikon,Tokyo, Japan) connected to a Nikon Digital Sight camera. Neutrophils per field were counted by Fiji ImageJ software (version 2.2.2-rc-34/1.50a, National Institutes of Health).
3
Transcriptomic Profiles of hiPS-Derived Endothelial Cells
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After sorting, 2.5–3.5 × 10^5 hiPS-ECs were plated on an Ibidi μ-Slide I Luer (80176, Ibidi). 4.0–6.0 × 10^5 hiPS-ECs were plated in one well in 6-well plate (static control). After 24 h, the cells on Ibidi slide were subjected to laminar shear stress of 15 dyn/cm2 by using the Ibidi Pump System (10902, Ibidi). After 24 h of exposure to flow, the cells were processed either for bulk RNA-sequencing or single-cell RNA-sequencing. The static control cells were processed at the same time. For bulk RNA-sequencing, the cells were collected into the RA1 lysis buffer and extracted using the Nucleospin RNA Plus Extraction kit (740984, Macherey-Nagel). Each experiment (whether for scRNASeq or bulk RNASeq) consists of one differentiation round from each hiPS cell line.
4
In vitro magnetic cell retention
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In vitro magnetic retention assays were carried out under flow conditions in a modified channel slide (μ-Slide I Luer, 0.4-mm height, ibidi) using a two-magnet system as previously described (43 (link)). The neodymium iron boron (NdFeB) permanent magnets (Supermagnete) used in this study were the following: an 5 × 14-mm magnet with 1.35 T of remanent magnetization (Br) (magnet A) and an 8 × 6-mm magnet with 1.45 T of remanent magnetization (Br) (magnet B). Briefly, either murine NK cells expanded in vitro in the presence of IL-2 or the human NK-92MI cell line (107 cells/ml) were treated with MNPs (150 μg Fe/ml) or not for 2 h in standard conditions, and then washed and stained with calcein-AM. An Olympus Inverted Microscope (model IX71) coupled to a Cell∧R imaging station under standard culture conditions was used in this assay. Shear stress was fixed at 0.5 dyne/cm2 and events were recorded every 1 s. After 60 s, the two-magnet system was applied for the next 2 min. Imaris software (Bitplane) was used to analyse displacement along the Y-axes (in the direction of the magnetic field).
5
Intracellular Lipid Droplet Spectroscopy
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U2OS cell line (human bone osteosarcoma epithelial cells) and Huh-7 cell line (human hepatocellular carcinoma cells) were supplied from Korean Cell Line Bank (Seoul, Korea) and cultured in DMEM (Welgene, LM 001-17) supplemented with penicillin (100 U mL−1), streptomycin (100 μg mL−1), 10% heat-inactivated fetal bovine serum (Welgene, S1010-01) at 37 °C and pH 7.4 (5% CO2). For IP measurements, the cells were subcultured with a cell density of 100 cells per mm2 in μ-Slide I Luer (ibidi, #80181), whose bottom is a borosilicate glass (170 μm in thickness) coated with 0.2 mg mL−1 poly-d -lysine (Sigma-Aldrich, P7886). To obtain IP spectra of individual LDs, cells were fixed using a fixation buffer containing 4% paraformaldehyde (Biolegend, #420801). The cells were treated with the fixation buffer for 10 minutes and then washed three times with phosphate-buffered saline (Welgene, LB 004-02).
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