Airway epithelial cell isolation and flow cytometry was performed as previously described by Farin et al.15 (link). Briefly, 5,000 sorted EpCAM+,CD31/34/45−,7AAD− epithelial cells were mixed with 100,000 unirradiated immortalized mouse fibroblasts. The mixture was added to an equal volume of growth factor reduced Matrigel (BD Biosciences) and seeded to the apical surface of 24-well transwell filter inserts (BD Biosciences) placed in 24-well flat-bottom culture plates. The solution was allowed to polymerize for 30 min at 37 °C, then basic medium was added to the basal compartment of the well. Cell cultures were maintained for 14 days at 37 °C in a humidified incubator (5% CO2). Colony-forming efficiency was calculated as the percentage of seeded cells that give rise to colonies, imaged on a Zeiss Axiovert40 fluorescent microscope and quantitated using FIJI. Enrichment for CD24med, Sca-1− distal and CD24med, Sca-1+ proximal airway epithelial cells was performed as previous described16 (link).
Axiovert40 fluorescent microscope
Manufactured by Zeiss
Sourced in Germany
The Axiovert40 is a fluorescent microscope designed for routine observation and documentation of fluorescently labeled samples. It features a stable stand, high-intensity illumination, and optical components optimized for fluorescence imaging.
Automatically generated - may contain errors
2 protocols using axiovert40 fluorescent microscope
1
Airway Epithelial Cell Isolation and Culture
2
Immunofluorescence Analysis of C2C12 Myotubes
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C2C12 myotubes were fixed in PBS containing 4% paraformaldehyde and permeabilized with PBS 0.5% triton X‐100. The preparation was incubated with an anti‐desmin primary antibody (1/100; Sigma‐Aldrich) diluted in PBS/BSA for 1 h at 37°C then washed in PBS, followed by a 30 min incubation with fluorescein‐conjugated anti‐mouse (1/100; Interchim Fluoprobes 488). Nuclei were stained with Hoechst (0.1 mg/mL; Sigma). The slides were examined with an Axiovert 40 fluorescent microscope (Carl Zeiss, Oberkochen, Germany). To estimate myotube size, the diameter of at least 500 myotubes per condition in at least three independent cultures was measured using the ZEN lite software (Carl Zeiss, Oberkochen, Germany). The average diameter per myotube was calculated as the mean of three measurements taken along the length of the myotube. The fusion index is defined as the proportion of cells that contain three or more nuclei. The fusion index was determined after 5 days of differentiation by counting at least 1000 nuclei per condition and per culture in three independent cultures.
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