The bovine serum albumin (BSA)-stabilized IL-4 MDs was fabricated according to previous studies.18 (link),19 (link),100 ng/mL IL-4 (R&D system, China) was used based on a previous study.20 (link) Briefly, 100 ng/mL IL-4, 300 μL perfluorocarbon, and 4 mL of PBS, and 40 mg of bovine serum albumin (BSA, Sigma Aldrich, China) were used for MDs fabrication. The resulting emulsion was ultracentrifuged (Beckman Coulter, Optima XPN-100, USA) at 14,000 rpm for 30 min, and the MDs-IL4 were used for subsequent testing. To generate fluorescence-labeled MDs for fluorescence imaging, fluorescein isothiocyanate (FITC)-labelled BSA (Sigma Aldrich, China) was used for MDs fabrication. Fluorescence-labeled MDs-IL4 were used for MDs characterization using a confocal laser scanning microscopy (CLSM). Briefly, 50 mg MDs-IL4 were diluted into PBS and then observed using a confocal laser scanning microscope with a ×40 objective (Leica DM IRB; Leica, Wetzlar, Germany). MDs-IL4 growth and rupture were observed using an inverted light microscope (Leica, Wetzlar, Germany). Briefly, the samples were exposed to an ultrasound probe with an acoustic frequency 1 MHz by portable home use ultrasound pain therapy device for 25 min (MYCHWAY, China). Images were captured using an inverted light microscope (Leica, Wetzlar, Germany).
Inverted light microscope
Manufactured by Leica
Sourced in Germany, United Kingdom
The Leica inverted light microscope is a versatile instrument that allows the observation and examination of specimens from an inverted perspective. It is designed to provide high-quality images and facilitate detailed analysis of samples, particularly those that require a specialized viewing angle or configuration.
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Lab products found in correlation
Matrigel, by BD (8 mentions)
Crystal violet, by Beyotime (4 mentions)
Crystal violet, by Merck Group (3 mentions)
Transwell chamber, by Corning (3 mentions)
Paraformaldehyde (pfa), by Solarbio (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Matrigel, by Solarbio (2 mentions)
Matrigel, by Corning (2 mentions)
Gentamycin, by Thermo Fisher Scientific (2 mentions)
Oil red o staining, by Merck Group (2 mentions)
Alcian blue stain, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Bovine serum albumin (bsa), by R&D Systems (1 mentions)
Dm irb, by Leica (1 mentions)
Interleukin 4 (il 4), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Interleukin 4 (il 4), by R&D Systems (1 mentions)
Optima xpn 100, by Beckman Coulter (1 mentions)
Colorimetric assay kit, by BioAssay Systems (1 mentions)
100 protocols using inverted light microscope
1
Fabrication and Characterization of Ultrasound-Responsive IL-4 Microdroplets
2
Transwell Invasion Assay for H1299 and H460 Cells
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Matrigel (364262, BD Biosciences) was coated on a 24-well transwell chamber (Costar; Corning, Inc.) at 37°C and placed in a cell incubator overnight. After suspending transfected H1299 cells and H460 cells with FBS-free medium, 100 μL of cells were seeded into the upper chamber at 5 × 105 cells/ml, and 500 μL of RPMI-1640 containing 10% FBS was added to the lower chamber. H460 cells were treated with or without the addition of 10 μM U0126. After culturing at 37°C for 24 h, cells on the underside of the membrane were fixed with 5% glutaraldehyde for 30 min at 4°C, and then stained with 0.5% crystal violet for 30 min at room temperature. Images were taken using an inverted light microscope (Leica, Germany) and invading cells in different areas were counted and analyzed using ImageJ software [29 (link)]. All experiments were repeated three times.
3
Liver Tissue Function Biomarkers
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Albumin and urea production as well as lactate dehydrogenase (LDH) release were measured before the drug-metabolism study (4 days postseeding). Albumin production was measured in supernatant using a human albumin enzyme-linked immunosorbent assay (Assay Pro, St. Charles, MO). Urea was quantified with a colorimetric assay kit (BioAssay Systems, Hayward, CA) and LDH secretion was measured using the CytoTox 96 nonradioactive cytotoxicity assay (Promega, Southampton, UK). Albumin, urea, and LDH were also measured postdose at the end of the drug-metabolism study (day 5 for wells treated with phenacetin; day 6 for wells treated with diclofenac, propranolol, lidocaine, and ibuprofen; and day 7 for wells treated with prednisolone). At the end of the experiment, the scaffolds/tissues were removed and washed with phosphate-buffered saline. Bright field images were taken using an inverted light microscope (Leica, Milton Kaynes, UK).
4
Transwell Invasion Assay for Cell Migration
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Cell invasion was assessed by transwell migration assays. Transwell chamber units were incubated with Matrigel Matrix (Corning, USA) at 37 °C for 1 h to generate an artificial basement membrane, and then rehydrated with 10 µL of serum-free RPMI 1640 medium. The upper chambers of the inserts were seeded with 4×104 cells stably transfected with control-shRNA, shRNA-ANXA7, or shRNA-JNK as indicated in 100 µL serum-free RPMI 1640 medium, whilst the lower chambers were filled with 500 µL of RPMI 1640 medium containing 20% FBS as a chemoattractant. After 48 h of incubation at 37 °C under a humidified 5% CO2 atmosphere, the transwell inserts were swabbed on the upper chamber side to remove non-invasive cells, fixed, stained with crystal violet, and then examined under an inverted light microscope (Leica, Japan) to assess cell numbers on the lower chamber side. Cell numbers in 5 randomly chosen high-power fields were counted per insert as an index of cell migration.
5
Defined Endothelial Cell Spheroid Sprouting
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Endothelial cell spheroids of defined cell number were generated59 (link). In brief, HUVECs and HPMECs were suspended in culture medium containing 0.20% (w/v) methylcellulose (MO262; Sigma-Aldrich, Taufkirchen, Germany) and seeded by using the hanging drop method. Spheroids were formed within 24 h with a defined cell number (400 cells/ spheroid and were embedded into collagen (#08-115, Millipore, Temecula, CA, USA). The spheroid-containing gel was rapidly transferred into prewarmed 96 well plate (Sarstedt, Nümbrecht, Germany) and allowed to polymerize for 30 min prior adding conditioned media on top of the gel. After 24 h, pictures of the sprouting spheroids were taken with an inverted light microscope (Leica, Wetzlar, Germany). Sprouting was quantified by counting the number of the sprouts that had grown out of each spheroid using Fiji v1.5.2, with 5 spheroids analyzed per experimental group and experiment.
6
Quantifying Cellular Morphology via Microscopy
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A divided Petri dish is plated with a single cell solution of ~2000 cells/ml and is incubated in standard lab conditions overnight to let the cells adhere in the surface of the dish. Accordingly, brightfield images of attached single cells are captured in 40x magnification and known acquisition parameters to an inverted light microscope (Leica, Germany). To check size and shape homogeneity between each cell population so that to assure that the estimated average cell size will be representative, we capture a photograph of a single cell solution within the fixed grid dimensions of the hemocytometer.
7
Oncogenic Transformation Assay in MEFs
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Wildtype mouse embryonic fibroblasts (MEFs) were harvested from C57BL/6J mice and underwent in vitro electroporation of SBKP, mLama4 SKBP, or mLama4/mItgb1 SBKP plasmids using the Lonza 4D-Nucleofector per the manufacturer’s instructions. Soft agar assays were used to evaluate cell line transformation potential as previously described19 (link). Briefly, 15,000 cells were plated in 0.4% bactoagar RPMI with 10% FBS and penicillin/streptomycin over a 0.6% bactoagar RPMI layer in 6 cm plates. Plates were incubated for 3 weeks to allow colony formation prior to staining with 0.05% crystal violet in 10% ethanol for 1 hour. Colonies were imaged using a Leica inverted light microscope and colonies were manually counted by an investigator blinded to treatment group.
8
Soft Agar Clonogenic Assay
9
Efficient Rabbit Embryo Genome Editing
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Rabbit embryos were obtained from diestrous female rabbits at an age of 6–8 months. These females were superovulated with 60 IU follicle stimulating hormone (FSH) at every 12 h (for six iterations) from 8 AM to 8 PM (60 IU in total). At the last time injection with FSH, the donors and recipients were injected with 10 IU human chorionic gonadotropin (HCG) simultaneously, and then, the donors were mated with fertile male rabbits. At 18–20 h later, embryos were flushed out using PBS and collected for microinjection. A mixed solution containing Cas9 mRNA (40 ng/ul)and multiple sgRNAs (13 ng/ul) was microinjected into the cytoplasm of embryos under a Leica inverted light microscope. The injected embryos were transferred into M2 cushion fluid and incubated at 38 °C, 5% CO2 for 30 min. 20–30 injected zygotes were transferred into the oviducts of pseudo-pregnant females.
10
Soft Agar Colony Formation Assay
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Colony formation in soft agar was used to determine cells’ capacity to colonize in in-vitro. A total of 1×103 cells of ZR75 were placed in RPMI medium containing 0.2% agar with/without drug(s) (treated and control cells, respectively) and plated in a 6-well plate covered with a layer of 0.4% noble agar in RPMI complete growth media (1 ml solid agar layer/well). A volume of 500 µl of media without (control) or with drug(s) were added to each well on 12th and 14th day of plating for ZR75 to make sure that the agar does not dry. The concentration range for BMS-202 was set to 1-20 µM, as our preliminary experiments on ZR75 colonies revealed no significant drug effect when treated with lower concentrations. Similar ranges were reported in (IC50 15 μM, in PD-L1+ SCC-3 cells and IC50 10 μM, in anti-CD3 activated Jurkat cells) (39 (link)), (0.6 nM up to 20 µM) (32 (link)), and (2.5-80 µM) (36 (link)) for various experiments based on different cell-lines. Colony formation was monitored every two days for a period of three weeks, and pictures of the colonies were taken on the 5th, 7th,9th, 12th, 14th, 17th, and 19th day after seeding from various locations in each well using the inverted light microscope (Leica, Germany).
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