RA-FLSs (4 × 103 cells per well) were seeded in eight-well chambers (Thermo Fisher Scientific) and grown to 80% confluence for 24 h. Recombinant ORM2 (1 μg/mL; Prospec-Tany Technogene) was added to each well and incubated at 37 °C for 1 h. The cells were then fixed with formaldehyde, permeabilized with 0.1% Triton X-100 for 3 min at room temperature, and incubated with an anti-ORM2 Ab (bs-7565R, 1:100; Bioss, Woburn, MA, USA) and/or an anti-GYPC Ab (sc-59183, 1:100; Santa Cruz Biotechnology) for 1 h at 37 °C. These cells were then stained with a Duolink In Situ Orange Starter Kit Mouse/Rabbit (Sigma‒Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Approximately 50–100 cells per area (at a magnification of 100×) were observed, and more than 5 randomly selected areas were subjected to analysis.
Eight well chamber
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Eight-well chamber is a laboratory equipment used for cell culture and sample preparation. It provides a contained environment with eight separate wells for performing parallel experiments or assays. The core function of the Eight-well chamber is to enable controlled and consistent sample handling and observation.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Bs 7565r, by Bioss Antibodies (1 mentions)
Duolink in situ orange starter kit mouse rabbit, by Merck Group (1 mentions)
Aqua poly mount mounting medium, by Polysciences (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Leica sp5 confocal microscope, by Leica Microsystems (1 mentions)
Bcecf am, by Thermo Fisher Scientific (1 mentions)
Bcecf am, by Abcam (1 mentions)
Ab7970, by Abcam (1 mentions)
A21206, by Thermo Fisher Scientific (1 mentions)
Lsm 800, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by BD (1 mentions)
6 protocols using eight well chamber
1
ORM2 Interaction with GYPC in RA-FLS Cells
2
Quantifying DNA Damage Response Foci
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Cells were seeded in eight-well chambers (Thermo Fisher Scientific) and treated as indicated in the figure legends. To stain for FIRRM-V5, RAD51, or RPA foci in cells, slides were pre-extracted with 0.2% Triton X-100 in PBS for 2 min on ice to remove nonchromatin bound protein and washed with PBS twice. Cells were fixed with 2% PFA or ice-cold 100% methanol (for RPA staining in KP) for 10 min. After permeabilization for 1 hour in immunofluorescence (IF) buffer (0.2% Triton X-100 and 10% FCS in PBS), slides were incubated with primary antibodies in IF buffer for 1 hour at room temperature. Cells were then washed three times and incubated with secondary antibodies (Invitrogen) and DAPI (2.5 μg/ml; Sigma-Aldrich) for 1 hour. Then, slides were washed again and mounted in Aqua-Poly/Mount mounting medium (Polysciences). Images were taken on a Leica SP5 confocal microscope (Leica Microsystems) or ZEISS LSM 980 Airyscan. Quantification of DNA damage–induced foci was performed in Fiji using CLIJ2/x libraries (48 (link)) with a custom ImageJ macro (v1.3; https://github.com/BioImaging-NKI/Foci-analyzer ; last accessed on 10 February 2023). Briefly, nuclei are recognized in the DAPI channel. DNA damage foci are detected by applying marker-controlled watershed (49 (link)) on local maxima in the foci channel, after which foci numbers and intensities are quantified per cell.
3
Co-localization of TREK1 and Rab5a in CHO-K1 cells
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In eight-well chamber (Thermo), CHO-K1 cells were co-transfected with PEGFP-N1-TREK1 and OFP-Rab5a. After 24 h, cells were treated with 10 μM TKDC for 20 min at room temperature and DMSO as control. At the end of incubation, cells were washed with PBS, fixed with 4% paraformaldehyde and washed with PBS. Images were taken at × 100 with scan zoom 1.74 using Leica SP8 and 3D reconstruction was done using Imaris software (Bitplane) and co-localization analysis was quantified using Imaris software. An automatic threshold of red and green channels was selected. The data represent images taken from 18 different areas (n = 18).
4
Monocyte-HUVEC Adhesion Assay
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THP-1 cells were labeled with the fluorescent molecule BCECF-AM (5 μmol/L, Abcam, UK) for 30 min at 37 °C, followed by washing twice with phosphate-buffered saline (PBS) and resuspension in RPMI-1640 medium. HUVECs were seeded in an eight-well chamber (Thermo Fisher Scientific, USA) and were pre-treated with 100 μg/mL of THD for 1 h. The confluent HUVEC monolayer was then stimulated with 100 μg/mL of oxLDL and incubated with BCECF-AM-labeled THP-1 cells (2 × 105/well) in RPMI-1640 medium containing 10% FBS at 37 °C for 30 min. Unbound monocytes were subsequently removed by washing twice with warm PBS.
5
Intracellular Localization of NF-κB in RA-FLSs
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To determine the intracellular localization of NF-κB, immunofluorescence staining of p65 (ab7970; Abcam, Cambridge, England) in RA-FLSs was performed. In brief, RA-FLSs (1 × 105 cells per well) were seeded into an eight-well chamber (Thermo Fisher Scientific) and grown to 80% confluence for 24 h. Recombinant human ORM2 (1 μg/mL; Prospec-Tany TechnoGene Ltd., Rehovot, Israel) was added to each well and incubated for 1 h. Cells were fixed with formaldehyde for 10 min and permeabilized with 0.1% Triton X-100 for 3 min at room temperature. After blocking with 10% normal donkey serum at room temperature for 1 h, the cells were stained with an anti-p65 Ab (ab7970, 1:100; Abcam) and Alexa 488-conjugated donkey anti-IgG (a21206, 1:1000; Thermo Fisher Scientific). After washing with PBS three times, the nuclei were stained with DAPI (1:500; BD Biosciences, San Jose, CA, USA). The stained cells were visualized with a confocal microscope (Zeiss, LSM800, Gottingen, Germany).
6
Multiplex RNA FISH Protocol for DANCR and MALAT1
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The RNA fluorescence in situ hybridization was carried out using the ACDBio RNAscope multiplex fluorescence assay kit. RNA probes targeting DANCR (NR_024031.2) and MALAT1 (NR_002819.4) were synthesized by ACDBio Company. FISH was performed according to the manufacturer’s instructions. Briefly, cells cultured in an eight-well chamber (Thermo Fisher Scientific, USA) were fixed in 4% PFA for 15 min at room temperature, and then treated with hydrogen peroxide solution and Protease III for 10 min, respectively. MALAT1 probes (C2, Opal 520) were diluted 1:50 in DANCR probe (C1, Opal 570) and pipetted into each well. Probe hybridization took place at 40 °C for 2 h and cells were rinsed in 1× wash buffer. Cells were sequentially incubated in AMP1 for 30 min, AMP2 for 30 min and AMP3 for 15 min at 40 °C, rinsed with 1× wash buffer between each incubation, for signal amplification. Cells were also counterstained with DAPI to visualize nuclei. Samples were subsequently observed with Zeiss LSM 880 confocal microscope.
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