Whole peripheral blood mononuclear cells (PBMCs) were extracted using Ficoll separation method. PBMCs were washed once with PBS, pelleted and were divided into two fractions. This step was repeated for both the viremic and the aviremic phases individually in order to assay p24 antigen-stimulated and un-stimulated fractions. Equal amount of cells were re-suspended for each fraction in RPMI medium 1640 containing 10% FBS in a non-adherent “Nunc® dishes, low cell binding” to yield the maximum amount of cells. One fraction of PBMCs was stimulated with HIV capsid protein p24 at 2 ng/mL. This is the standard concentration used for assaying IFNγ production using Enzyme-linked immuno-sorbent spot (ELISPOT) assay. Both the un-stimulated and stimulated fractions were incubated overnight at 37 °C with 5% CO2.
Nunc dishes
Manufactured by Thermo Fisher Scientific
Sourced in Germany, Austria
Nunc dishes are cell culture dishes designed for the growth and observation of cells in a laboratory setting. They provide a stable and controlled environment for cell cultivation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Upcell surface, by Thermo Fisher Scientific (2 mentions)
Laminin, by BD (2 mentions)
Poly d lysine, by Merck Group (2 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Infinity m200 plate reader, by Tecan (1 mentions)
Penicillin g, by Merck Group (1 mentions)
Streptomycin sulfate, by Merck Group (1 mentions)
Celltiter glo reagent, by Promega (1 mentions)
Amphotericin b, by Merck Group (1 mentions)
Transwell plate, by Corning (1 mentions)
Mg 63, by American Type Culture Collection (1 mentions)
Gentamicin, by Ratiopharm (1 mentions)
A1rmp image system, by Nikon (1 mentions)
Nis elements software v4, by Nikon (1 mentions)
A1r scanner, by Nikon (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
Fluorodeoxyuridine, by Merck Group (1 mentions)
13 protocols using nunc dishes
1
PBMC Separation and Stimulation Protocol
2
Cell Growth Dynamics Quantification
3
Antifungal Fiber Mats Cytotoxicity Assay
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Both the antifungal-loaded and bare fiber mats were adhered to microscope cover slips (n=4) and plated onto 24-well Nunc dishes. Human corneal and sclera fibroblasts were grown in HyClone® Dulbecco’s Modified Eagle’s Medium (Thermo Fisher) with 10% fetal bovine serum antimycotic solution containing 500 units/mL penicillin G, 0.1 mg/mL streptomycin sulfate, and 2.25 μg/mL amphotericin B (Sigma-Aldrich). The cells were seeded at a concentration of ~4×104 cells/well and incubated with the fiber mats at 37°C for 24 hours. After equilibrating the plates to room temperature, an equal volume of CellTiter-Glo® reagent was added (Promega Corporation, Madison, WI, USA). The adenosine triphosphate content was determined by recording the luminescence on a Tecan Infinity M200 plate reader as per the manufacturer’s instruction.
4
Boar Sperm-Oocyte Fertilization Protocol
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Following IVM, COCs were placed in a 100 μl drop of HEPES-199 supplemented with 0.5 mg/ml hyaluronidase for 1
min, and gently aspirated in a fresh HEPES-199 droplet using a fine bore glass pipette to partially remove
cumulus cells. Partially denuded oocytes were placed in TALP-PVA media in 4-well Nunc dishes (~50 oocytes per
500 µl well). A straw of frozen boar semen was thawed at 42 C in a water bath for 20 sec and the sperm
solution was centrifuged at 720 g at 37 C for 10 min. The sperm pellet was then retrieved,
and resuspended in TALP-PVA medium before being centrifuged again at 310 g at 37 C for 5 min.
The sperm pellet was again resuspended in TALP-PVA medium and sperm concentration and motility were assessed.
To each well, a total of 250 motile sperm per oocyte was added. After 30 min, oocytes and zona-bound sperm
were carefully moved to a second well containing 500 µl TALP-PVA medium and incubated for a further 5 h at
38.5 C in a humidified atmosphere of 6% CO2 in air.
min, and gently aspirated in a fresh HEPES-199 droplet using a fine bore glass pipette to partially remove
cumulus cells. Partially denuded oocytes were placed in TALP-PVA media in 4-well Nunc dishes (~50 oocytes per
500 µl well). A straw of frozen boar semen was thawed at 42 C in a water bath for 20 sec and the sperm
solution was centrifuged at 720 g at 37 C for 10 min. The sperm pellet was then retrieved,
and resuspended in TALP-PVA medium before being centrifuged again at 310 g at 37 C for 5 min.
The sperm pellet was again resuspended in TALP-PVA medium and sperm concentration and motility were assessed.
To each well, a total of 250 motile sperm per oocyte was added. After 30 min, oocytes and zona-bound sperm
were carefully moved to a second well containing 500 µl TALP-PVA medium and incubated for a further 5 h at
38.5 C in a humidified atmosphere of 6% CO2 in air.
5
Porcine RPE Cell Isolation and Cultivation
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Porcine RPE cells were isolated as previously described [13 (link)]. Briefly, porcine eyes obtained from the local abattoir were cleaned of adjacent tissue and immersed briefly in antiseptic solution (betaisodona, Mundipharma). The anterior part of the eye was removed, as well as the lens, vitreous, and retina. In each eyecup, trypsin was added, and incubated for 10 min at 37 °C. The trypsin solution was removed and substituted with trypsin-EDTA for 45 min at 37 °C. RPE cells were gently pipetted off the choroid, collected in media, and washed. Cells were cultivated in DMEM supplemented with penicillin/streptomycin (1%), HEPES (25 mM), sodium-pyruvate (110 mg/ml), 10% fetal calf serum (Linaris GmbH, Wertheim-Bettingen, Germany), and 1% non-essential amino acids. Cells of the second passage were cultivated on Transwell plates (volume of medium: 200 µg apical, 600 µl basal; Costar Corning), and the transepithelial resistance (TER) was measured with an epithelial voltmeter (EVOM; World Precision Instruments, Sarasota, FL) as previously described [23 (link)]. For western blot experiments, RPE cells were cultured on Nunc dishes (Thermo Scientific, Schwerte, Germany).
6
Osteoblastic Cell Culture Protocol
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Samples were washed in 70% ethanol for 15 min, rinsed in phosphate-buffered saline (PBS) (PAA Laboratories, Pasching, Austria) and then placed into 4-well NUNC dishes (Thermo Fisher Scientific, NUNC GmbH & Co. KG, Langenselbold, Germany). Afterwards human osteoblastic cells (MG-63, purchased from ATCC; No. CRL-1427) were seeded at a density of 3×104 cells/array in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen GmbH, Karlsruhe, Germany), containing 10% fetal calf serum (FCS) (PAA Laboratories, Pasching, Austria) and 1% gentamicin (Ratiopharm GmbH, Ulm, Germany) at 37°C in a humidified atmosphere with 5% CO 2.
7
In Vivo Spinal Cord Injury Transplantation
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For in vivo transplantation, Mφs were isolated from the same mouse strain as the recipient mice in the in vivo experiment, being either C57BL/6J or BALB/c. Mφs were cultured using Nunc™ Dishes with UpCell™ Surface (ThermoFisher Scientific) to avoid enzymatic harvesting. Immediately after SCI induction, mice were randomized and received an intraspinal injection of vehicle or (GFP+) Mφs. As previously described, a motorized stereotaxic injector pump (Stoelting) with a 10 µl Hamilton syringe (34-gauge needle) was positioned 1–3 mm rostral to the lesion [19 ]. The needle was stereotactically inserted into the spinal cord at a depth of 1 mm. Either vehicle (PBS or RMPI) or cells (1 × 104 in 2.5 µl) were injected over a 4 min period, after which the needle was kept in place for again 4 min to allow pressure equilibration and prevent backflow. Investigators were blinded to the treatment groups during the complete experiment.
8
Isolation and Culture of Cortical Neurons
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Cortical neurons were dissected from E15.5 C57BL/6J mouse embryos and dissociated as above. Neurons were plated on Nunc dishes (Thermo Fisher Scientific) or glass coverslips coated with 40 μg/mL poly-D-lysine (Sigma) and 2 μg/mL Laminin (BD Bioscience) in MEM supplemented with 10% fetal bovine serum, 5% horse serum, 1 mM glutamine and 1× penicillin-streptomycin. After 2–6 h, culture medium was replaced with Neurobasal medium supplemented with 1× B27, 1 mM glutamine, 1× penicillin-streptomycin and 10 μM FdU (Merck). Cells were cultured at 37°C, 5% CO2 for 2–7 days.
9
Confocal and Live-Cell Imaging of Cells
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Microscopy of fixed cells was performed using a laser-scanning confocal microscope Nikon A1RMP image system with a Nikon A1R scanner, Nikon A1-DUG 4 channel filter based detector unit and Nikon LUNV Laser Launch (6 lines) with a X60 water immersion objective. Cells plated on glass coverslips were washed twice with PBS and fixed with 4% paraformaldehyde post-transfection. Images were processed using Nikon NIS-Elements software v4.5. The excitation/emission wavelengths during acquisition were 488 nm/492–557 nm for YFP
For live imaging, cells were plated on Nunc® dishes (ThermoFisher Scientific). Images and time-lapse videos were captured on a Nikon Eclipse Ti-E Inverted Fluorescent fluorescence microscope image system with an Andor Zyla sCMOS Monochrome camera, Lumencor Spectra X Light Engine and Tokai-Hit Stage Incubation chamber with 40X and 20X objective. Images were collected every 30 s for 24 hours and then processed identically between each condition to adjust brightness and contrast using the Nikon NIS-Elements software v4.5. Each experiment was repeated 3 times, and 5–7 fields were visualized in each experiment.
For live imaging, cells were plated on Nunc® dishes (ThermoFisher Scientific). Images and time-lapse videos were captured on a Nikon Eclipse Ti-E Inverted Fluorescent fluorescence microscope image system with an Andor Zyla sCMOS Monochrome camera, Lumencor Spectra X Light Engine and Tokai-Hit Stage Incubation chamber with 40X and 20X objective. Images were collected every 30 s for 24 hours and then processed identically between each condition to adjust brightness and contrast using the Nikon NIS-Elements software v4.5. Each experiment was repeated 3 times, and 5–7 fields were visualized in each experiment.
10
Uniaxial Cyclic Tensile Stimulation System
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A custom system was built for applying uniaxial, cyclic tensile mechanical stimulation (Fig. 2 ). The system houses 4 separate culture chambers to enable simultaneous stimulation of multiple samples of cells. The culture chamber is a disposable, sterile, rectangular culture plate (Thermo Scientific Nunc Dishes, 267061, Waltham, Mass.). Silicone elastomer substrates (polydimethylsiloxane, PDMS) were added into the culture chambers as a stretchable surface for applying mechanical strain. An actuating stage applies the strain to the cells.
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