Confluent HBE cells in cell-culture treated glass bottom microtiter plates (Greiner bio-one) were stained with the CellTracker Orange CMTMR dye (5 μM, excitation 548/emission 576, ThermoFisher Scientific, Cat# C2927) in serum-free MEM for 30 min. The medium was replaced with 100 μl MEM without phenol red (ThermoFisher/Gibco) and cells were treated with USA300 supernatants and liposomes (300 μg/ml) or sodium Tyrode's buffer (vehicle) for 1 h. Imaging was performed with a ZEISS LSM 800 confocal microscope equipped with an incubation system at 37 °C and analyzed with the ZEISS ZEN software 2.3 blue edition.
Mem without phenol red
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
MEM without phenol red is a cell culture medium formulated for the growth and maintenance of various cell lines. It is a variation of the original Minimum Essential Medium (MEM) formulation, with the key distinction being the absence of the pH indicator dye phenol red. This allows for more accurate colorimetric measurements and analyses that may be affected by the presence of phenol red.
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Lab products found in correlation
Lsm800 confocal microscope, by Zeiss (2 mentions)
Celltracker orange cmtmr dye, by Thermo Fisher Scientific (1 mentions)
Zen 2.3 blue edition software, by Zeiss (1 mentions)
Eclipse ti, by Nikon (1 mentions)
Nis element, by Nikon (1 mentions)
Chopstick electrode, by World Precision Instruments (1 mentions)
Evom epithelial voltohmmeter, by World Precision Instruments (1 mentions)
D luciferin, by Merck Group (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Flutamide, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rankl, by Thermo Fisher Scientific (1 mentions)
Leukocyte acid phosphatase kit, by Merck Group (1 mentions)
Penicillin, by Beyotime (1 mentions)
Streptomycin, by Beyotime (1 mentions)
M csf, by R&D Systems (1 mentions)
5 protocols using mem without phenol red
1
Staining and Imaging of Confluent HBE Cells
2
Live/Dead Cell Fluorescence Assay
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Two solutions were selected for the live/dead analysis. A modified Eagle’s medium (MEM) without phenol red (Gibco®, Gaithersburg, MD) was used for this experiment. Briefly, cells were seeded in three 8-chamber slides (Lab-Tek®) at a concentration of 1.5 × 104 cells/well and were then incubated for 24 hrs to allow cell adhesion to occur. The culture medium in each well was replaced with 300 μl of each solution and then incubated for 2 hrs. Finally, 10 μl of EB/AO fluorescent dye was added to each chamber, and the fluorescence of the cells was analyzed under a fluorescent inverted microscope (ECLIPSE Ti, Nikon, Tokyo, Japan), with 10X magnification. Images were captured with imaging software (NIS-Elements, Nikon, Tokyo, Japan). The EB/AO fluorescent dye was prepared by mixing 15 mg acridine orange with 50 mg ethidium bromide powder that was first dissolved in 1 ml 95% ethanol and then diluted in 49 ml of distilled water (dH2O).
The images were evaluated by two evaluators according to previously described criteria. Under the green filter, an intact green nucleus, which is comparable to the control, indicates a viable cell, whereas a green fragmented nucleus indicates early apoptosis. A red nucleus indicates a ruptured cell wall, whereas a red intact nucleus indicates necrosis, and a fragmented red nucleus indicates late apoptosis under the red filter [27 (link),28 (link)].
The images were evaluated by two evaluators according to previously described criteria. Under the green filter, an intact green nucleus, which is comparable to the control, indicates a viable cell, whereas a green fragmented nucleus indicates early apoptosis. A red nucleus indicates a ruptured cell wall, whereas a red intact nucleus indicates necrosis, and a fragmented red nucleus indicates late apoptosis under the red filter [27 (link),28 (link)].
3
Measuring Cell Layer Integrity via TEER
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The trans-epithelial electrical resistance (TEER) measurement was used to confirm cell layer integrity (Figure 1 D). Cell culture inserts were apically filled with 350 µL MEM without phenol red (Gibco®, Invitrogen, Schwerte, Germany) and the abluminal/basolateral compartment filled with 500 µL MEM. With incubation for 20 min at 37 °C and 15 min at room temperature (RT), the cells were allowed to equilibrate to the new conditions. The TEER values were determined in triplicate using an EVOM epithelial voltohmmeter and chopstick electrodes (World Precision Instruments, Friedberg, Germany). The raw data were processed by subtracting the blank (inserts without cells) and by multiplying with the area of the insert’s membrane (0.336 cm2). The cut-off values for OEPC and RPMI 2650 confluent cell layers were determined as described previously [36 (link)].
4
Quantitative Virus Infection Assay
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HEp-2 cells were seeded at 5 × 104 cells per well in 96-well plates the day before, and infected with serially threefold-diluted viral suspensions beginning with 3,000–10,000 p.f.u. per well. For the mCherry assay, MEM without phenol red (Gibco) was used for cell culture and virus dilutions. For the luciferase assay, infected cells were incubated at 37 °C and 5% CO2 for 24 h, then washed in 1 × phosphate-buffered saline (PBS) before lysis with 50 μl of lysis buffer (25 mM Tris pH 7.8, 8 mM MgCl2, 1% Triton X-100, 15% glycerol, 1 mM dithiothreitol) per well for 15 min at room temperature with gentle shaking. Plate and substrate were equilibrated at 37 °C before luciferase activity measurement, expressed in RLU using a Tecan infinite M200PRO plate reader and automatic injection for delivering 100 μl of 2 μM D -luciferin (Sigma) and ATP 10 mM in lysis buffer. For mCherry fluorescence measurement, infected cells were incubated at 37 °C and 5% CO2 for 48 h. Fluorescence could be observed with a fluorescence microscope and was measured using a spectrofluorometer (Tecan infinite M200PRO) with excitation and emission wavelengths of 580 and 620 nm, respectively, (expressed in relative fluorescence units). Non-infected HEp-2 cells were used as standards for fluorescence and luminescence background levels.
5
Osteoclastogenesis Modulation Assay
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Minimum essential medium (MEM) without phenol red and fetal bovine serum (FBS) were obtained from Gibco-BRL (Gaithersburg, MD, USA). Penicillin, streptomycin, and a kit for the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). M-CSF was supplied by R&D Systems (Minneapolis, MN, USA). RANKL was obtained from Peprotech (Rocky Hill, NJ, USA). DHEA, E2, flutamide, and a Leukocyte Acid Phosphatase Kit were purchased from Sigma-Aldrich Co (Saint Louis, MO, USA). MPP and R,RTHC were obtained from Tocris Cookson Inc. (Ellisville, MO, USA). An active DHEA enzyme immunoassay (EIA) kit was purchased from Diagnostic System Laboratories, Inc. (DSL, Webster, Texas, USA). A DHEAS EIA kit was supplied by IBL (Fujioka, Japan). An E2 EIA kit was supplied by BioCheck Inc. (Burlingame, CA, USA).
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