To determine the effect of TQ on association of TRAF-2 and ASK1 in TNF-α signaling, RA-FLS (2 × 104 cells/well) were plated in 8-well chamber slides (Corning, NY). At 70% confluence, cells were starved overnight and pretreated for 2 hrs with or without TQ 5 μM followed by stimulation with 20 ng/ml TNF-α for 30 min. Cells were washed twice with ice cold PBS and fixed in 3% paraformaldehyde PBS solution for 10 min at room temperature. Cells were washed 3 time with ice cold 1X PBS and stored in serum free RPMI for overnight. Cells were permeabilized by lysis buffer (20 mM PIPES-pH 6.8, 100 mM NaCl, 3 mM MgCl2, 1 mM EDTA, and 0.5% triton X100) for 10 min at room temperature followed by washing with PBS. Coverslips were blocked using 5% protease free BSA for 1 hrs at room temperature. Primary antibodies mouse monoclonal TRAF-2 (1:250) or rabbit polyclonal ASK1 (1: 100) were used at room temperature for 2 hrs, followed by 3 washes using 1% BSA PBS. Donkey anti mouse Alexa Flour 594 or Donkey anti rabbit Alexa Flour 488 (Invitrogen, CA) as 1:400 dilution were used as secondary staining for 60 min at room temperature. After 3 washes in 1% BSA PBS slides were mounted using Prolong gold anti-fade with DAPI (Invitrogen, CA). Images (40X magnification) were captured using Leica microscope.
8 well chamber slide
Manufactured by Corning
Sourced in United States
The 8-well chamber slides are a laboratory equipment designed to facilitate cell-based experiments. These slides provide a convenient platform for culturing cells and performing various analyses. Each slide contains 8 individual chambers, allowing for multiple experimental conditions to be tested simultaneously. The chambers are constructed with durable materials and are compatible with standard microscopy techniques.
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Lab products found in correlation
Prolong gold antifade with dapi, by Thermo Fisher Scientific (2 mentions)
Bz x800 microscope, by Keyence (2 mentions)
Donkey anti mouse alexa flour 594, by Thermo Fisher Scientific (1 mentions)
Mrc 1024, by Bio-Rad (1 mentions)
Formaldehyde, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (1 mentions)
Aggrecan, by Merck Group (1 mentions)
B27 supplement, by Thermo Fisher Scientific (1 mentions)
Poly d lysine pdl, by Merck Group (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant containing dapi, by Thermo Fisher Scientific (1 mentions)
Formaldehyde, by Thermo Fisher Scientific (1 mentions)
Dylight 554 phalloidin, by Cell Signaling Technology (1 mentions)
Rabbit anti cd31, by Novus Biologicals (1 mentions)
Dmi4000b fluorescence microscope, by Leica (1 mentions)
Evos xl core cell imaging system, by Thermo Fisher Scientific (1 mentions)
Las x, by Leica (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
Vectasheild with dapi, by Vector Laboratories (1 mentions)
Cytopainter phalloidin ifluor 594 reagent, by Abcam (1 mentions)
21 protocols using 8 well chamber slide
1
Investigating TQ's Effect on TRAF-2 and ASK1
2
DAPI Staining for Apoptosis Assay
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DNA staining was performed using 4',6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich) for morphological observation of apoptotic cells [26 (link)]. HCT116 (7 × 103 cells/well) and H1299 (6 × 103 cells/well) cells were seeded on 8-well chamber slides (Corning Inc.). After 24 h, cells were treated with PLE at 0, 87.5, 175, and 350 µg/ml in serum free media for 24 h. Cells were fixed using 4% formaldehyde (Sigma-Aldrich) for 15 min at room temperature, followed by incubation with DAPI. The nuclear morphology was observed using confocal laser scanning microscopy at a magnification of 200 fold (MRC-1024, Bio-Rad Laboratories). Apoptotic cells were recognized by condensed, fragmented, or degraded nuclei [27 (link),28 (link)]. DAPI staining intensity was determined using Image-J software (NIH, Bethesda, MD, USA).
3
Culturing Primary Mouse Cortical Neurons
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Embryos of ddY mice (Japan SLC, Japan) were obtained at the 14th day of gestation. The dura was removed, and cerebral cortices were isolated, minced, and dispersed. Cells were cultured on 8-well chamber slides (Corning, NY), coated with poly-D-Lysine (PDL; 5 μg/mL; Sigma-Aldrich, St. Louis, MO), at a density of 1.5 × 104 (link) cells/well, in Neurobasal medium (Thermo Fisher Scientific, Waltham, MA) containing 12% horse serum, L-glutamine, and 0.6% D-glucose at 37°C in a humidified incubator with 10% CO2. Five hours after seeding, the medium was replaced with fresh Neurobasal medium containing 2% B-27 supplement (Thermo Fisher Scientific), 0.6% D-glucose, and 2 mmol/L L-glutamine. For CSPG coating, culture dishes were coated with 5 μg/mL PDL (Sigma-Aldrich) and 2 μg/mL aggrecan (Sigma-Aldrich) in Hank's buffered salt solution (HBSS; Thermo Fisher Scientific) overnight at 37°C.
4
Tyramine-Induced Norepinephrine Release
5
Fluorescent Visualization of A549 Cells
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A549 cells were seeded (1 × 104) on 8-well chamber slides (Corning, 354630). A day after cells were fixed by using 4% formaldehyde (Thermo Fisher Scientific, 28906) for 15 min at RT, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 3% BSA (Cell Signaling Technology, 9998) for 1 h. DyLight™ 554 phalloidin (Cell Signaling Technology, 13054) was used to stain F-actin (1:200 dilution in PBS) for 15 min at RT. Chamber slides then incubated with Prolong Gold Antifade Mountant containing DAPI (Thermo Fisher Scientific, P36931).
6
Isolation and Characterization of Mesenchymal Stem Cells
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Some scaffolds explanted at 4 weeks (n = 2) after implantation were used. They were reduced in small pieces and digested at 37 °C for 2 h in PBS containing collagenase type I (0.5 U/ml). The suspension was filtered, suspended in mesenchymal stem cells growth medium (Lonza Group Ltd.) and plated on 8-well chamber slides (Corning) at a density of 5000 cells/cm2. The medium was changed twice/week until reaching 70–80% confluence. Then, cells were fixed with 4% paraformaldehyde and stained by using the following antibodies: mouse anti-CD73 (1:25, Novus Biologicals); rabbit anti-CD105 (1:50, Novus Biological), rabbit anti-CD31 (1:50, Novus Biological) and rabbit anti-CD45 (1:100, Epitomics). Briefly, slides were blocked for 1 h at room temperature (RT) with 5% normal donkey serum and 0.3% Triton-X100 in PBS and incubated for 2 h, at RT with the primary antibodies. Then, sections were processed for the secondary incubation with the appropriate Alexa Fluor 568 antibody (Life Technologies) at the dilution of 1:2000. After washing, slides were counterstained with DAPI (1:10 000), mounted and examined by using a Leica DMI 4000B fluorescence microscope (Leica Microsystems).
7
Visualizing Uropathogenic E. coli Interaction with Urothelial Cells
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RT4 cells were seeded onto 8‐well chamber slides (Corning) at 3 × 104 cells per well and grown to confluence. Cells either preconditioned or not with HA (2 mg mL−1) were challenged for up to 24 h with TPA4935J, a clinical UPEC isolate transformed with pEGFp‐C3 (102 per well). After washing with PBS, fixing in 4% PFA the cells were stained for 2 h with CytoPainter Phalloidin‐iFluor 594 reagent (Abcam) diluted 1:1000, and coverslips mounted using Vectasheild with DAPI (Vector Laboratories). Images were captured using a Leica SP8 confocal microscope (fluorescence) or an EVOS XL Core Cell Imaging System (Thermo Fisher; light microscope images). Image analysis was conducted using LAS X (Leica) and Image J software.
8
Isolation and Culture of Human Nasal Epithelial Cells
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Primary HNECs were harvested from nasal mucosa by gentle brushing. Extracted cells were suspended in PneumaCultTM- Ex Plus medium (StemCell Technologies, Vancouver, Canada).
Macrophages were removed by treating the cells with anti-CD68 (Dako, Glostrup, Denmark) coated petri dishes for 20 min at 37 ℃. HNECs were maintained with PneumaCultTM- Ex Plus medium in collagen coated flasks (Thermo Scientific, Walthman, MA, USA) at 37 °C with 5% CO2 until confluence. 5*105 cells were seeded in collagen coated 24 well plates (Corning, NY, USA) and 8 well chamber slides (Corning, NY, USA) respectively. Cells were treated with 5% bacterial planktonic supernatants for different times followed by RNA extraction, immunofluorescence staining or protein extraction as described below. Cells treated with purified SpA (50 μg /ml) and 5% TSB were used as positive and negative control, respectively.
Macrophages were removed by treating the cells with anti-CD68 (Dako, Glostrup, Denmark) coated petri dishes for 20 min at 37 ℃. HNECs were maintained with PneumaCultTM- Ex Plus medium in collagen coated flasks (Thermo Scientific, Walthman, MA, USA) at 37 °C with 5% CO2 until confluence. 5*105 cells were seeded in collagen coated 24 well plates (Corning, NY, USA) and 8 well chamber slides (Corning, NY, USA) respectively. Cells were treated with 5% bacterial planktonic supernatants for different times followed by RNA extraction, immunofluorescence staining or protein extraction as described below. Cells treated with purified SpA (50 μg /ml) and 5% TSB were used as positive and negative control, respectively.
9
Immunofluorescent Staining of Active Caspase-3
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MP2 cells were grown to partial confluence in 8-well chamber slides (Corning) in DMEM with 10% fetal calf serum and were treated 24 h as stated. Then they were fixed by 4% paraformaldehyde, permeabilized using 0.25 Triton, stained using the indirect technique with rabbit anti-caspase-3 (active) (Genetex, Irvine, CA) at 1:200 dilutions followed by ALEXA-fluor-donkey anti-rabbit 568 (InVitrogen, Carlsbad, CA) at 1:200 dilutions and FITC-phalloidin (Sigma, St. Louis, M0) at 1:500 dilution, mounted using Prolong Gold antifade with DAPI (In Vitrogen, Carlsbad, CA). The slides were examined using Olympus B-max fluorescence microscope at 20× and 40× magnification with the Hamamatsu camera, and fluorescence was determined in four fields using Image Pro software and expressed as Integrated Optical Density (IOD) per macrophage.
10
Platelets Adhesion Assay with COVID-19 Serum
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For the assessment of the adhesion ability of platelets, an adhesion assay was performed. In brief, after incubation of platelets with COVID‐19 ICU or control serum, platelets (3 × 106 platelets/well) were seeded in 8 well chamber slides (Corning) that were pre‐coated with 100 µg/ml of fibrinogen (Sigma Aldrich) overnight. Platelets were allowed to rest for 1 h at RT before staining. TRAP‐6 (10 µM; Hart Biologicals) was used as a positive control. Where indicated, platelets were preincubated with inhibitors BAY and BYL as previously described. In further experiments, GPIIb/IIIa was blocked by 10 min of pre‐incubation at 37°C, with eptifibatide (2.4 µM, accord; Middlesex). Afterwards, slides were fixed with 2% paraformaldehyde (PFA; Morphisto) for 20 min at RT in the dark. For adhesion quantification, images were captured from five randomly chosen microscopic fields and analyzed with the CellSens Standard software (Olympus) (×60; Olympus IX73).
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