The MLO-Y4 cell line was provided by Dr. Lynda Bonewald (University of Missouri, Kansas City, MO, USA) (28 (link)). The cells were cultured in MEM (Gibco, Paisley, UK) with 2.5 percent fetal bovine serum (FBS) and 2.5 percent calf serum including 1 percent penicillin/streptomycin. Cells (5×105 cells/dish) in the logarithmic phase were seeded in 35 mm culture dish (Nunc, Inc. Roskilde, Danemark) for 24 hrs, and then treated by irrelevant rabbit IgG antibody (CWBIO, Beijing, China, 1:50 dilution) in an incubator at 37°C with 5 percent CO2 for 30 mins, then separated into 4 groups: control (C), 1g, 2g and μg groups (Table 1 ). The cells of C group were cultured in the same incubator for another 3 hrs, and at the meantime the cells of 1g, 2g and μg groups were placed on sample holder and cultured on the HMGE platform with temperature controlled at 37±0.5.°C and 5 percent CO2 for 3 hrs (15 (link)). For antibody treated group, Cx43(E2) antibody (1:50 dilution, 28 μg/ml) was added into the media in 35 mm culture dish for 30 min, the identical culture condition with previous study (22 (link)), and then separated into 4 groups: E2-C, E2-1g, E2-2g and E2-μg groups (Table 1 ). Cells of E2-1g, E2-2g and E2-μg groups were cultured in the given positions on HMGE platform, while cells of E2-C group were cultured in the same incubator with C group.
35 mm culture dish
Manufactured by Thermo Fisher Scientific
Sourced in China
The 35-mm culture dish is a common laboratory equipment used for cell culture. It provides a contained and controlled environment for the growth and observation of cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Minimum essential medium (mem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
D glucose, by Merck Group (1 mentions)
Medium 199, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Sodium bicarbonate, by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Merck Group (1 mentions)
Neurobasal a, by Thermo Fisher Scientific (1 mentions)
Lumicycle, by Actimetrics (1 mentions)
Lumicycle analysis software, by Actimetrics (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Mz125, by Leica (1 mentions)
L aspartate, by Merck Group (1 mentions)
Turbofect transfection reagent, by Thermo Fisher Scientific (1 mentions)
Pegfp n1, by Thermo Fisher Scientific (1 mentions)
6 protocols using 35 mm culture dish
1
Gravity Effect on MLO-Y4 Osteocyte Cells
2
Circadian Rhythm Monitoring in Retinal Explants
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Mice were killed by cervical dislocation 1 h before light offset (Zeitgeber Time 11 or ZT11), except the Opn4−/−::rd/rd::Per2Luc mice that were euthanized at CT11 according to their behavioral locomotor rhythm. Eyes were enucleated and placed in Hank’s balanced salt solution (HBSS; Invitrogen) on ice. Retinas were gently isolated from the rest of the eye cup and flattened ganglion cell layer upon a semipermeable (Millicell) membrane in a 35-mm culture dish (Nunclon) containing 1.2 mL Neurobasal-A (Life Technologies) with 2% B27 (Gibco), 2 mM L-Glutamine (Life Technologies), and 25 U/mL antibiotics (Penicillin/Streptomycin, Sigma), incubated at 37 °C in 5% CO2 for 24 h. From this step on, all manipulations of explants were performed under dim red light. After 24 h, at the projected ZT12, retinas were transferred to 1.2 ml of 199 medium (Sigma), supplemented by 4 mM sodium bicarbonate (Sigma), 20 mM D-glucose (Sigma), 2% B27, 0.7 mM L-Glutamine, 25 U/mL antibiotics (Penicillin/Streptomycin, Sigma), and 0.1 mM Luciferin (Perkin). Culture dishes were sealed and then placed in a Lumicycle (Actimetrics, Wilmette, IL, United States of America) to record the global emitted bioluminescence. For blocking gap junction, 100 μM CBX was added to 199 medium. PER2::Luc bioluminescence was analyzed using Lumicycle Analysis software (Actimetrics, Wilmette, IL, USA).
3
Visualizing bPNA-dye Uptake in HEK-293T Cells
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HEK-293T cells were cultured based on ATCC protocol. Cells were seeded to a 35-mm culture dish (Thermo Fisher) with a clean coverslip (Thermo Fisher) attached to the bottom at a concentration of 5 × 105/ml. After one day of incubation, the cells were transfected with corresponding plasmids (1000 ng each per dish) with Lipofectamine 3000 (Invitrogen) following the online protocol, and the cells were incubated for 24 h. Culture medium was removed and the fresh medium containing 1 µM bPNA-dye was added to the attached cells. The cells were incubated at 37 °C for 2–8 h, medium discarded, and Hoechst 33258 (Invitrogen) in fresh culture medium was added at the final concentration of 200 ng/ml to the cells and incubated for 15 min at 37 °C. Cells were washed with PBS, fixed with 4% formaldehyde in PBS for 15 min at room temperature and rinsed with PBS. The coverslip was transferred to glass slides for fluorescence microscopy and imaged under Olympus FV3000 systems (Objective lens: ×40, Zoom: ×1. Blue channel: (excitation) 405 nm, (emission) 422 nm, Voltage: 355 V, detection range: 420–470 nm; Green: (excitation) 488 nm, (emission) 520 nm, Voltage: 580 V, detection range: 499–599 nm). Three or more replicate data sets were obtained that showed consistent findings, starting from cell seeding.
4
Larval Zebrafish Eye Physiology Recordings
5
Murine in vitro Fertilization and Embryo Transfer
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Collected COCs were placed in 100 μl drops of TYH medium (LSI Medience Corporation) covered by mineral oil in 35 mm culture dish (Thermo Fisher Scientific). These dishes were placed into CO2 incubator before sperm introduction for fertilization. After COCs collection, mature male ICR mice were immediately sacrificed and epididymis was removed for sperm preparation. This epididymis was punctured with a 21 G needle, and the sperms were squeezed into equilibrated 600 μl TYH medium. The sperms were stored in CO2 incubator for 10 min, and then, sperm suspension was added to the drops of TYH medium which contained COCs to give a final sperm concentration of 5.0 × 104/ml. At 6 h after insemination, zygotic stage embryos were collected from the cumulus cells by gently pipetting and cultured in 30 μl of EmbryoMax® KSOM mouse embryo medium (Merck Millipore) covered with mineral oil at 37°C and 5% CO2 in air up to specific stage embryos.
Female ICR mice (6–8 weeks of age) were mated with a vasectomized male ICR mouse to produce pseudo‐pregnant mice as a recipient for embryo transfer. Ten to sixteen blastocysts were transferred into the uterus of the day 3.5 pseudo‐pregnant recipients in each experimental group.
Female ICR mice (6–8 weeks of age) were mated with a vasectomized male ICR mouse to produce pseudo‐pregnant mice as a recipient for embryo transfer. Ten to sixteen blastocysts were transferred into the uterus of the day 3.5 pseudo‐pregnant recipients in each experimental group.
6
Measuring ANO1-Mediated Current in HEK293T Cells
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To measure the ANO1-mediated current, HEK293T cells were transfected with human ANO1 (hANO1) expression vector using TurboFect transfection reagents (Thermo Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. The ratio of green fluorescent protein (pEGFP-N1, Life Technologies) to hANO1 vector (10:1) was used to confirm the successful transfection of the cells. HEK293T cells were prepared the previous day in a 35-mm culture dish (Thermo Fisher Scientific). Then, 1.8 mg of hANO1 vector, 0.2 mg pEGFP, and 4 ml of TurboFect were added to 200 μl of DMEM (without FBS and P/S), followed by incubation at 25°C for 15 min. Lastly, 2 ml of complete medium (DMEM, 10% FBS, and 1% P/S) was added to the mixture and cultured with the prepared HEK293T cells for 24 h.
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