MTT and BrdU incorporation assays were performed as previously described [24 (link),14 (link)]. Cells were seeded in 96-well plates at 1 × 104 cells per well. Colony formation was determined by using 3D soft gar matrix with a 0.8% of soft agar (noble agar) base layer followed by 0.4% top agar layer containing cells at a density of 1 × 104 with different treatments in 24-well culture plates. Colonies were counted using inverted light microscopy. For transmigration assay, the ability of migration and invasion of cells was examined using BD BioCoat Matrigel Invasion Chambers (BD Biosciences) according to manufacturer’s instruction. After 24 h, the migrated cells were pelleted and counted using a hematocytometer. To further confirm cell migration, a wound healing assay was performed in 12 well plates (1 × 105 per well). When the cells were grown to 90 to 95% confluences, tranfection of CD133+ cells with siRNA-snoRA42 and scrambled siRNA, as well as mock transfection were performed. Wound lines were created manually by scratching the monolayer with a sterile 200 ml pipette tip and migration of the cells was assessed after 24 hours. Pictures were taken using Nikon inverted phase-contrast microscope (Nikon, Melville, NY). The distance between the parallel lines was measured using ImageJ software. All experiments were carried out at least three times.
Inverted phase contrast microscope
Manufactured by Nikon
Sourced in Japan, United States
The Inverted Phase Contrast Microscope is a laboratory instrument designed to observe transparent specimens. It uses phase contrast illumination to enhance the visibility of otherwise invisible structures within the sample. The core function of this microscope is to provide high-contrast images of living cells, tissues, and other translucent samples.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by BD (11 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Dexamethasone (dex), by Merck Group (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Matrigel, by Corning (2 mentions)
Metamorph, by Molecular Devices (2 mentions)
Ultra low attachment 6 well plate, by Corning (2 mentions)
Matrigel, by Nikon (2 mentions)
Digital camera, by Nikon (2 mentions)
Trypsin, by Thermo Fisher Scientific (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Dexamethasone (dex), by Solarbio (2 mentions)
Ascorbic acid, by Solarbio (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Biocoat matrigel invasion chamber, by BD (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Gfr matrigel, by BD (1 mentions)
μ angiogenesis slides, by Ibidi (1 mentions)
129 protocols using inverted phase contrast microscope
1
Evaluating Cell Proliferation and Migration
2
3D Cell Aggregate Sprouting Assay
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The hanging drop method was applied to obtain 3D aggregates of cells.29 Briefly, 24 h treated cells were suspended in a mixture of 70% proper culture medium, 10% FBS (Sigma‐Aldrich) and 20% methylcellulose. Drops of 25 µl of the suspension (2000–3000 cells/drop) were distributed equally over a 10 cm petri dish. The plates were incubated upside down overnight at 37°C (5% CO2, 95% humidity) to allow formation of a single stable spheroid. The next day, all hanging drops were collected into a 50 ml falcon tube and embedded into 1.5% rat tail collagen gels (cat. no. 354236; Corning). To prepare the collagen solution according to manufacturer's instruction, we mixed, 3% collagen with an equal volume of 0.85% (wt/vol) methylcellulose in culture medium with 10% FBS (Sigma‐Aldrich). The spheroid suspension was pipetted into 24‐well plates (350 µl/well), and, incubated for 30 min. Then, the polymerized collagen gels were overlaid with appropriate culture medium containing 0.5% FBS and, incubated at 37°C, 5% CO2 with 95% humidity. ImageJ software and Nikon inverted phase‐contrast microscope were used to determine cumulative sprout length per spheroid. For each experiment at least five spheroids were evaluated.
3
Hemocyte Monitoring in Bat Torpor
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The hemocyte populations of active (n = 6), mid-torpor (n = 6) and fully torpid (n = 6) colonies were isolated from the vasculature (Avishai et al., 2003 (link)). Specimens were individually drained into a small petri dish, and each was stained with a fluorescent dye Hoechst 33342 (cat: 62249, Thermo Fisher Scientific) for nuclei staining. The cells were then washed several times with filter seawater (to remove excess dye) and monitored daily with a Nikon inverted phase contrast microscope for 10 days periods.
4
Microscopy and Cytometry Protocol
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The following instruments were used: inverted phase-contrast microscope, biological microscope (Nikon, Japan), electrophoresis tank, flow cytometer, CO2 constant temperature incubator, plate reader, centrifuge, and JJ260 precision electronic balance (American BD company).
5
Analyzing HUVEC Tube Formation on Matrigel
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Growth factor-reduced (GFR) Matrigel (BD Bioscience) was thawed on ice at 4 °C overnight. Ten microlitres of Matrigel was plated onto each inner well of the μ-angiogenesis slides (IBIDI, Germany) and allowed to solidify at 37 °C for 30 min. 1 × 104 HUVECs were reconstituted in CM only, CM containing anti-VEGF neutralizing antibody (10 μg/ml), CM with IgG isotype control (10 μg/ml), complete medium, EGM or SFM to a total volume of 50 μl and plated on the GFR Matrigel. The plates were incubated for 6 h in a humidified incubator at 5% CO2 and 37 °C. Images were taken using an inverted phase-contrast microscope (Nikon) under 4× and 10× objectives. The tube length was measured using WimTube (Wimasis, GmbH, Germany) from the 4× magnification images of three wells for each condition. The experimental samples and controls were assayed in triplicates.
6
SH-SY5Y Protection Against H2O2
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SH-SY5Y cells were seeded into 6-well culture plates at a density of 1×105 cells/ml. Cells were treated with TPC at concentrations of 0.5 and 5 μg/ml for 1 h before exposing them to 200 μM H2O2. The cellular morphology was observed using inverted phase contrast microscope (Nikon, Tokyo).
7
Proliferation of Inner Ear-Derived Cells
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After culturing on the MEF-ECM and TCP for 1, 4,
and 7 days, the morphology of inner ear-derived cells
was observed by an inverted phase contrast microscope
(Nikon, Japan). For proliferation assays, the CCK-8
(Sigma, USA) was employed to detect the proliferation
of inner ear-derived cells at P4 and P8 cultivated on
the MEF-ECM and TCP. The inner ear-derived cells
at a density of 1×104cells per well were seeded on the
MEF-ECM and TCP with DMEM/F12 supplemented
with 10% FBS and 50 ng/mL ampicillin at 37˚C under
5% CO2atmosphere. After culturing for 1, 4 and 7 days,
CCK-8 working solution was introduced into the wells.
After incubation for a duration of 2 hours at 37˚C, optical
density (OD) values were taken at 450 nm by using a plate
reader (Thermo Scientific, Waltham, MA, USA).
and 7 days, the morphology of inner ear-derived cells
was observed by an inverted phase contrast microscope
(Nikon, Japan). For proliferation assays, the CCK-8
(Sigma, USA) was employed to detect the proliferation
of inner ear-derived cells at P4 and P8 cultivated on
the MEF-ECM and TCP. The inner ear-derived cells
at a density of 1×104cells per well were seeded on the
MEF-ECM and TCP with DMEM/F12 supplemented
with 10% FBS and 50 ng/mL ampicillin at 37˚C under
5% CO2atmosphere. After culturing for 1, 4 and 7 days,
CCK-8 working solution was introduced into the wells.
After incubation for a duration of 2 hours at 37˚C, optical
density (OD) values were taken at 450 nm by using a plate
reader (Thermo Scientific, Waltham, MA, USA).
8
Isolation and Characterization of Murine Splenocytes
9
Morphological Effects of UA on NRK-52E Cells
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The NRK-52E cells were seeded in equal amounts (4×105 cells per well) into 6-well plates and then exposed to UA (0, 50 and 100 µg/ml) for 24 h. After the cell-drug interaction, the growth of NRK-52E cells in each group was observed under an inverted phase contrast microscope (Nikon Corporation) to investigate the morphological changes (magnification, ×400).
10
MCF7 Cell Viability Assay with MEAD
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MCF7 cells were cultured in 24-well tissue culture plates (Beckton, Dickinson, Franklin Lanes, NJ) at a seeding density of 2×104 cells/well. After overnight attachment, the cells were treated with different concentrations (5, 10, 15, 20 and 25 mg/ml) of MEAD and the untreated cells served as control (S1 Methods ). The cells were cultured for up to 72 h under standard culture conditions of 37°C in a humidified atmosphere of 95% air and 5% CO2. Changes in cell morphology were imaged using inverted phase contrast microscope (Nikon Instruments, Tokyo Japan).
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