Cells were seeded onto 8-well NuncTM Lab-TekTM (ThermoFisher Scientific, Madrid, Spain) and grown and treated as previously described and cells were fixed with 4% PFA for 10 min. Afterwards, cells were permeabilized with 0.5% Triton ×100 in PBS for 5 min. Non-specific binding was blocked by incubation with 3% BSA (Sigma-Aldrich, Madrid, Spain) for 30 min at room temperature. Incubation with a specific primary antibody was carried out overnight with ZO-1 rabbit polyclonal antibody 1/500 (Thermo Fisher Scientific, Madrid, Spain). Samples were rinsed with PBS and subsequently incubated for 1 h at room temperature with Alexa-488 donkey anti-rabbit secondary antibody (LifeTechnologies, Oregon, USA) at a dilution of 1/800 and Hoechst (Sigma-Aldrich, Madrid, Spain) at 1/2000 v/v. Images were acquired in a confocal microscope (TCS SP5, Leica, Wetzlar, Germany).
Nunc lab tektm
Manufactured by Thermo Fisher Scientific
Sourced in Spain, United Kingdom
The Nunc™ Lab-Tek™ is a laboratory equipment product designed for cell culture applications. It provides a multi-well format with a variety of well sizes and surface treatments to accommodate different experimental needs. The product serves as a platform for performing various cell-based assays and analyses.
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Lab products found in correlation
Tcs sp5, by Leica (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Hoechst, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Normal horse serum and casein, by Vector Laboratories (1 mentions)
Eclipse 90i microscope, by Nikon (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Poly d lysine, by Merck Group (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Laminin, by Thermo Fisher Scientific (1 mentions)
Uridine, by Merck Group (1 mentions)
Fluorodish fd35 100, by World Precision Instruments (1 mentions)
5 fluoro 2 deoxyuridine, by Merck Group (1 mentions)
4 protocols using nunc lab tektm
1
Immunofluorescence analysis of tight junctions
2
Phagocytosis of Diesel Exhaust Particles
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Phagocytosis of carbonaceous diesel exhaust particles (DEP) in vitro by AM was assessed by adhering AM overnight in Nunc TM Lab-Tek TM cell culture chambers (ThermoFisher Scientific, UK) precoated with collagen (Sigma-Aldrich, Missouri, USA). Adherent AM were then incubated with 10 μg/mL DEP (SRM 2975, NIST, USA; collected from diesel-powered forklifts) for 2 h, followed by cytocentrifugation and imaging under light microscopy for AM carbon area (above).
In some experiments, adherent AM from controls and children with CF were incubated with 10 -6 M PGE 2 (P0409, Sigma-Aldrich, USA) , a concentration that inhibits phagocytosis of carbonaceous PM by human monocyte-derived macrophages [20] . The effect of CF sputum supernatant on DEP phagocytosis was assessed using responder AM from healthy controls. To assess the effect of sputum supernatant from children with CF, sputum supernatant was diluted 1:1 with medium and added to wells before exposure of AM to DEP (10 μg/mL, as above). An EP2-receptor antagonist (75 μM, AH6089, Sigma-Aldrich, USA) was added in some experiments.
In some experiments, adherent AM from controls and children with CF were incubated with 10 -6 M PGE 2 (P0409, Sigma-Aldrich, USA) , a concentration that inhibits phagocytosis of carbonaceous PM by human monocyte-derived macrophages [20] . The effect of CF sputum supernatant on DEP phagocytosis was assessed using responder AM from healthy controls. To assess the effect of sputum supernatant from children with CF, sputum supernatant was diluted 1:1 with medium and added to wells before exposure of AM to DEP (10 μg/mL, as above). An EP2-receptor antagonist (75 μM, AH6089, Sigma-Aldrich, USA) was added in some experiments.
3
Endothelial Differentiation Markers in MSCs
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To identify the expression of relevant endothelial differentiation markers in ADSC, BMSC, and DPSC, we carried out immunofluorescence analyses using specific antibodies anti‐vascular endothelial growth factor (VEGF), anti‐CD31 (PECAM‐1), anti‐von Willebrand Factor (vWF), and anti‐CD34. These analyses were carried out on native, non‐differentiated, and differentiated MSCs. HUVEC was used as a control group. First, 104 cells were cultured in chamber slides (NuncTM Lab‐TekTM Thermo Fisher Scientific), fixed with 70% ethanol, washed in PBS, and blocked for 30 min with normal horse serum and casein (Vector Laboratories). Then, samples were incubated for 1 h with primary antibodies with a dilution 1:100 for anti‐VEGF (Abcam), 1:500 for anti‐CD31 (PECAM‐1; Abcam), 1:100 for anti‐vWF (Abcam), and 1:2500 for anti‐CD34 (Abcam), washed in PBS, and incubated with secondary antibodies for 1 h at room temperature using FITC‐conjugated anti‐mouse (Sigma‐Aldrich/Merck) or CY3‐conjugated anti‐rabbit antibodies (Sigma‐Aldrich/Merck). All study samples and controls were counterstained with DAPI (Vector Laboratories) and analyzed using an Eclipse 90i microscope (Nikon). Results were evaluated by three independent histologists using a semiquantitative method scoring the signal as strongly positive (+++), positive (++), mildly positive (+), slightly positive (+/−), or negative (−).
4
Dissection and Culture of DRG Neurons
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All plates for DRG cultures are coated with 0.1 mg/ml poly-D-lysine (Millipore Sigma) followed by laminin (3 µg/ml; Invitrogen). CD1 mouse and SARM1 KO DRG neurons were dissected from embryonic day 13.5 or 14.5. They were incubated with 0.05% trypsin containing 0.05% EDTA at 37 ºC for 20 mins and then washed 3 times with DRG growth medium (neurobasal media from Gibco) containing 2% B27 (Invtrogen), 50 ng/ml nerve growth factor (Harlan Laboratories), 1 µM 5-fluoro-2'-deoxyuridine (Millipore Sigma), 1 µM uridine (Millipore Sigma), and penicillin/ streptomycin (Thermo Fisher Scientific). The cell density of these suspensions was adjusted to ~7 x 10 6 cells/ml. 2 µl suspensions were placed in 24-well plates (Corning) for western blots and axon degeneration assays, Chamber slides (Nunc TM Lab-Tek TM , Thermo Fisher Scientific) were used for immunocytochemistry and FluoroDish (FD35-100, World Precision Instruments) were used for live single axon imaging. Lentivirus was transduced at 1 or 2 days in vitro (DIV). At DIV 7, assays for axon degeneration and/ or live axon imaging were performed.
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