MDMs were differentiated on glass coverslips as described above. For actin filament staining, the cells were incubated with 5 µM LPA species for 30 min, fixed with 4% paraformaldehyde for 10 min at room temperature, and permeabilized with 0.3% Triton X100 for 5 min. Actin filaments were stained with Phalloidin-California Red Conjugate (1:1000; AAT Bioquest, Sunnyvale, CA, USA) for 30 min at room temperature. Glass coverslips were mounted on microscope slides using mounting medium with DAPI (VEC-H-1200, Vector, Burlingame, CA, USA) and sealed with nail polish. Images were taken at 40× magnification and processed on a widefield microscope (Leica DM5500, Leica Microsystems, Wetzlar, Germany). Images of three random fields per sample were analyzed with imageJ 1.54 (https://imagej.net/ij/ ).
Vec h 1200
Manufactured by Vector Laboratories
Sourced in United States
The VEC-H-1200 is a high-performance centrifuge that can achieve a maximum speed of 12,000 RPM and a maximum relative centrifugal force of 20,000 x g. It is designed for a variety of laboratory applications that require efficient separation of samples.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using vec h 1200
1
Actin Filament Staining of Differentiated MDMs
2
Senescence-Associated β-Galactosidase Staining in HGPS Cells
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HGPS cells grown on coverslips were washed once in PBS, fixed in 2% formaldehyde/0.2% glutaraldehyde for 5 minutes, and then stained overnight at 37°C with a solution containing 1 mg/mL 5-bromo-4-chloro-3-inolyl-β-galactosidase in dimethylformamide (20 mg/mL stock), 5 mM potassium ferricyanide, 150 mM NaCl, 40 mM citric acid/sodium phosphate, and 2 mM MgCl2, at pH 6.0 [39 (link)]. The cells were then washed twice with PBS and further processed for indirect immunostaining using anti-α-tubulin. DNA was stained with DAPI (Vector Inc., VEC-H-1200). Images were acquired using an Axioskop 2 mot plus fluorescence microscope (Objective X20 and X50, Carl Zeiss). ImageJ was used to merge single channel images. Blue β-galactosidase staining is presented as black signal in the bright-field image.
3
Actin Filament Staining in Macrophage-Derived Cells
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MDMs were differentiated on glass coverslips as described above. For actin filament staining the cells were incubated with 50 µM AA for 24 h, fixed with 4 % paraformaldehyde for 10 min at room temperature and permeabilized with 0.3% Triton X100 for 5 min. Actin filament was stained with 1:1000 diluted Phalloidin-California Red Conjugate (AAT Bioquest, Sunnyvale, USA) for 30 min at RT. Glass coverslips were mounted onto the microscope slides using a drop of mounting medium with DAPI (VEC-H-1200, Vector, Burlingame, USA) and sealed with nail polish. Images were taken at 100× magnification and processed on a widefield microscope (Leica DM5500, Leica Microsystems, Wetzlar, Germany).
4
Macrophage STAT Activation Imaging
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Monocyte‐derived macrophages cultured on cover slips were treated with IFNγ or IL6 for 30 min after preincubation with AA 50 µm for 30 min. Cells were fixed with 3.7% paraformaldehyde for 10 min at room temperature and washed three times with PBS. Fixed cells were permeabilized with 0.2% Triton‐X100 for 5 min at room temperature and blocked with bovine serum albumin (BSA) blocking buffer (5% BSA in PBS+ 0.1% Tween 20) for 30 min at room temperature. The cells were stained with anti‐STAT1 or anti‐STAT3 antibody diluted in blocking buffer overnight at 4 °C and secondary antibody diluted in blocking buffer for 1 h at room temperature in the dark. Coverslips were mounted on to glass slides using a drop of mounting medium with 4′,6‐diamidin‐2‐phenylindol (DAPI; VEC‐H‐1200; Vector, Burlingame, CA, USA) and sealed with nail polish. Images were acquired by confocal microscopy (Leica SP8; Leica Microsystems, Wetzlar, Germany).
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