Mammalian cells and neurons were maintained at 37 °C in a humidified cell culture incubator with 5% CO2. Authenticated HEK293 and HeLa cell lines (both American Type Culture Collection) cells were purchased directly from the vendor and the lines were morphologically correct. HeLa and HEK293 cells were cultured in imaging dishes containing poly-D-lysine coated coverslip glass bottoms (P35GC-0-14-C, MatTek Corporation) in DMEM supplemented with 10% FBS, 20 U/mL penicillin and 50 μg/mL streptomycin. Cortical neurons were dissociated by papain from postnatal day 2 (P2) Sprague Dawley rats and cultured in imaging dishes containing poly-D-lysine coated coverslip glass bottoms (P35GC-0-14-C, MatTek Corporation) in Neurobasal A medium supplemented with 1X B27 Supplements (both from Life Technologies), 2 mM GlutaMAX (Life Technologies), 20 U/mL penicillin and 50 μg/mL streptomycin. All animal procedures were approved by the Institutional Animal Care and Use Committee of UC San Diego.
P35gc 0 14 c
Manufactured by MatTek
Sourced in United States
The P35GC-0-14-C is a cell culture dish designed for use with various microscopy techniques. It features a 35 mm diameter glass bottom and a polystyrene culture area of 8.95 cm2. The dish is suitable for live-cell imaging applications.
Automatically generated - may contain errors
Lab products found in correlation
Hek293, by American Type Culture Collection (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Genjet in vitro dna transfection reagent for mcf7 cells, by SignaGen (2 mentions)
Genjet in vitro dna transfection reagent for u2os cells, by SignaGen (2 mentions)
Dmem high glucose medium, by Thermo Fisher Scientific (2 mentions)
Accutase, by Corning (2 mentions)
Cary 5000 uv vis nir spectrophotometer, by Agilent Technologies (1 mentions)
Sc 53564, by Santa Cruz Biotechnology (1 mentions)
Lsm 5 pascal, by Zeiss (1 mentions)
Fetal bovine serum (fbs), by Nichirei Biosciences (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
En0521, by Thermo Fisher Scientific (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Dnase, by Thermo Fisher Scientific (1 mentions)
F7524, by Merck Group (1 mentions)
Tcs sp5 confocal laser scanning microscope, by Leica (1 mentions)
Fc010, by Merck Group (1 mentions)
Lsm 510 meta, by Zeiss (1 mentions)
12 protocols using p35gc 0 14 c
1
Culturing Mammalian Cells and Neurons
2
Culturing Mammalian Cells and Neurons
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Mammalian cells and neurons were maintained at 37 °C in a humidified cell culture incubator with 5% CO2. Authenticated HEK293 and HeLa cell lines (both American Type Culture Collection) cells were purchased directly from the vendor and the lines were morphologically correct. HeLa and HEK293 cells were cultured in imaging dishes containing poly-D-lysine coated coverslip glass bottoms (P35GC-0-14-C, MatTek Corporation) in DMEM supplemented with 10% FBS, 20 U/mL penicillin and 50 μg/mL streptomycin. Cortical neurons were dissociated by papain from postnatal day 2 (P2) Sprague Dawley rats and cultured in imaging dishes containing poly-D-lysine coated coverslip glass bottoms (P35GC-0-14-C, MatTek Corporation) in Neurobasal A medium supplemented with 1X B27 Supplements (both from Life Technologies), 2 mM GlutaMAX (Life Technologies), 20 U/mL penicillin and 50 μg/mL streptomycin. All animal procedures were approved by the Institutional Animal Care and Use Committee of UC San Diego.
3
Optical Properties of Melanized Yeasts
4
Immunofluorescence Imaging of Adrenal Medulla Cells
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After collagenase treatment, one or two pieces of adrenal medulla tissue were placed into a dish with non-fluorescent glass (P35GC-0-14-C: MatTek, Ashland, MA, USA) and then dissociated using fine needles. The dissociated AMC cells were fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS; pH 7.2) for 1 hr and then pre-incubated in PBS with 5% fetal bovine serum (FBS) (Nichirei, Tokyo, Japan) and 0.3% Triton X-100 for 30 min. For indirect immunofluorescence, the cells were incubated with mouse anti-caveolin-1 Ab (sc-53564: Santa Cruz) (RRID:AB_628859), rabbit anti-CaV1.2 Ab, and/or mouse anti-M4 Ab. After incubation, the cells were washed three times in PBS and then incubated with the appropriate secondary Ab conjugated to Alexa Fluor 488 or 546 (Molecular Probes). The fluorescence was observed using a confocal laser scanning microscopy (LSM5 Pascal, Carl Zeiss, Tokyo, Japan). An oil-immersion objective lens with a magnification of 63× and a numerical aperture of 1.4 was used. For Alexa Fluor 488, a 488 nm laser was used and emission was observed at 510–560 nm (FITC-like fluorescence), whereas for Alexa Fluor 546, a 543 nm laser was used and emission was observed above 560 nm (rhodamine-like fluorescence). Immunocytochemical examination was repeated at least three times to confirm the reproducibility of the immunoreaction.
5
Neonatal Mouse Brain Cell Isolation
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Neonatal mice (postnatal day 0–2) were sacrificed using Home Office-approved methods (proscribed by the Animals Act, 1986). Brains were dissected out and submerged in ice-cold dissection medium, consisting of DMEM:F12 (D6421, Sigma), 1% L-glutamine (A2916801, Gibco), and 1% Pen Strep (15140122, Gibco). The cortices of the brains were separated from the midbrain and cerebellum. The meninges were removed using fine forceps, and the brains were homogenized by trituration followed by digestion in trypsin (25200056, Gibco) and DNase (EN0521, Thermo Scientific) for 20 minutes at 37°C. Cells were diluted in DMEM (41965039, Gibco) with 10% FBS (F7524, Sigma), 1% Pen Strep (15070, Thermofisher) (serum medium), and centrifuged at 300 × g for 5 minutes at 10°C. Following resuspension of the pellet cells were seeded into poly-d -lysine coated imaging dishes (P35GC-0-14-C, Mattek) at a density of 5 × 105 cells. The cultures were then incubated at 37°C/8% CO2 for 16 hours to allow the cells to settle, followed by a complete media change. Cells were then cultured for 9–12 days, with a ⅔ media change (supplemented with 3 μg/mL insulin) performed every third day. Mixed glial cultures were used for imaging at 15 DIV.
6
Labeling and Imaging CXCR4+ U2OS Cells
7
Quantitative Analysis of F-Actin Dynamics
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The cells were seeded onto the glass-bottom of fibronectin (FC010, Millipore, Temecula, CA, USA) coated culture dishes (P35GC-014-C; MatTek, Ashland, MD, USA). Following the 36 h attachment, the cells were fixed with 3.7% paraformaldehyde, and then, permeabilized with 0.1% Triton X-100 in phosphate buffered saline (PBS) at room temperature for 10 min. The cells were washed with PBS and blocked in a 1% bovine serum albumin (BSA)/PBS solution for another 1 h. The F-actin protein expression was revealed by its incubation with Texas Red X-Phalloidin (Invitrogen) and the immunofluorescence was recorded using a confocal microscope (LSM510 Meta, Zeiss, Oberkochen, Germany), as described previously [20 (link)]. The F-actin fluorescence intensity was assessed using Zen blue edition software (version 3.2, Zeiss). The intensity profiles were measured along the line from the peripheral to the central of the cells.
8
Metabolic Perturbations on EV Secretion
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To examine the effect of metabolic perturbations of parent cells on their corresponding EVs, MDA-MB-231 cells were incubated with phenol-free, serum-free, and glutamine-free DMEM with 1% PSA, and either: 25 mM glucose, 0 mM glucose, 0 mM glucose + 5 mM sodium pyruvate (11360070, Gibco, Waltham, MA), 0 mM glucose + 5 mM sodium L-lactate (71781, Sigma-Aldrich, St. Louis, MO), or 25 mM glucose + 100 μM CoCl2 (ACROS Organics, Thermo Fisher Scientific, Waltham, MA). The first four conditions alter the energy metabolism of the cells whereas CoCl2 induces chemical hypoxia41 (link).
After reaching 70–90% confluence, three T175 flasks were designated to each condition. Cell were rinsed twice with sterile 1 × PBS and overlain with the corresponding supplemented serum-free DMEM for their metabolic condition. Cells were returned to the incubator for 48 h and isolated as previously described. On the same day, cells in alternate T175 flasks with DMEM and FBS were designated for cell imaging and were plated (48 h prior to imaging) on poly-d-lysine coated glass bottom dishes (MatTek Life Sciences, Ashland, MA; P35GC-0-14-C), with three dishes assigned to each treatment group. Approximately 24 h prior to cell imaging, complete cell media was removed and replaced with serum-free DMEM supplemented for the corresponding metabolic perturbation.
After reaching 70–90% confluence, three T175 flasks were designated to each condition. Cell were rinsed twice with sterile 1 × PBS and overlain with the corresponding supplemented serum-free DMEM for their metabolic condition. Cells were returned to the incubator for 48 h and isolated as previously described. On the same day, cells in alternate T175 flasks with DMEM and FBS were designated for cell imaging and were plated (48 h prior to imaging) on poly-d-lysine coated glass bottom dishes (MatTek Life Sciences, Ashland, MA; P35GC-0-14-C), with three dishes assigned to each treatment group. Approximately 24 h prior to cell imaging, complete cell media was removed and replaced with serum-free DMEM supplemented for the corresponding metabolic perturbation.
9
Cell Culture and Transfection Protocol
10
Cell Culture and Transfection Protocol
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HeLa, HEK293T, MCF7, and U2OS cells, purchased from ATCC, were grown at 37°C and 5% CO2 in DMEM high glucose medium (GIBCO, #11965092) supplemented with 10% FBS (Corning Cellgro, 35–010-CV), 1 mM sodium pyruvate (GIBCO, #11360070), and 1X nonessential amino acids (GIBCO, #11140076). Transient transfections were performed using GenJet In Vitro DNA Transfection Reagent ver. II (SignaGen, #SL100499), GenJet In Vitro DNA Transfection Reagent for MCF7 cells (SignaGen, #SL100489-MCF7), or GenJet In Vitro DNA Transfection Reagent for U2OS cells (SignaGen, #SL100489-OS) following the manufacturer’s instructions. For biosensor barcoding, 2×105 cells were seeded in 12-well plates and allowed to attach overnight. Cells in each well were transfected with a pair of barcoding proteins and one biosensor, using a total of 0.75 μg plasmid per well. The next day, cells were harvested from each well with Accutase (Corning, 25–058-CI) and mixed together. The resulting cell mixture was then seeded into 35 mm glass-bottom dishes (Mattek, P35GC-0–14-C) at 7×105 cells per dish and incubated at 37°C and 5% CO2 overnight. The cells were starved in serum-free, phenol red-free DMEM (GIBCO, #21063029) for 1 h before imaging experiments.
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