Evos digital inverted microscope

Human alveolar basal epithelial (A549) cells were propagated in Dulbecco's modified Eagle medium (DMEM; Life Technologies, Thermo Fisher Scientific) supplemented with 10% (vol/vol) fetal bovine serum (FBS, Thermo Fisher Scientific), 100 U/ml penicillin and 50 µg/ml streptomycin at 37°C in a humidified atmosphere of 5% CO₂. Cells were seeded at a density of 4 × 10⁴ cells/well and grown to approximately 60% confluency.
Inflammatory response was induced in half of the cell cultures by incubating cell monolayers with 0.39 mg/ml dexamethasone (Sigma) and 50 ng/ml human recombinant interleukin-6 (Cambridge Bioscience, UK) in DMEM supplemented with 10% FBS. After 48 hr, cells were chilled at 4°C for 30 min and then incubated with 1 mg/ml SfbI-A40-TED-iPT, or SfbI-A40-TED:Cys109Ala-iPT in PBS, or PBS for 30 min at 4°C. Cells were fixed with pre-chilled (−20°C) methanol for 20 min at −20°C, then stained with 1 mg/ml sIPD-GFP in PBS for 30 min at 25°C. Nuclei were stained with 0.5 µg/ml DAPI (15 min, 25°C). Between each incubation cell monolayers were washed extensively with sterile PBS. After 24 hr at 4°C, cells were imaged on an EVOS Digital Inverted Microscope (Life Technologies, Thermo Fisher Scientific).

Immunofluorescence staining was used to determine the effect of vancomycin on the differentiation of the isolated muscle cells. Therefore cells were cultured in collagen (Corning^®; Discovery Labware; Amsterdam; Netherlands) coated 24 well plates. After reaching 80% confluency the medium was replaced by differentiation medium including vancomycin in different concentrations. Five days later, cells were washed twice with PBS, followed by fixation with ice cold methanol (Carl-Roth^® GmbH, Karlsruhe, Germany) for 20 min. After a second washing step with PBS and 20 minutes incubation with the primary antibody skeletal muscle myosin (F59) anti mouse (1:200 in 0,5 % BSA/PBS; Santa Cruz Biotechnology, Texas, TX., USA) was added and incubated at 4 °C overnight. As secondary antibody Alexa 488 goat anti mouse IgG (1:200; Invitrogen™Life Technologies, Carlsbad, USA) was applied for 1 h in the dark followed by washing with PBS. Finally nuclei were stained with Hoechst (4 µg/mL; Sigma Aldrich, St. Louis, MO, USA) with an incubation time of 15 minutes at room temperature in the dark. Detection was performed with the EVOS^® digital Inverted Microscope (Life Technologies, Carlsbad, CA, USA).

U2OS-GFP -α-tubulin cells (5 × 10⁵/well) were seeded into 6-well plates, each including a sterile ibi Treat μ-slide (ibidi, Germany). The cells were allowed to attach overnight, treated with 40 µM of SAF or DMSO (solvent control) and incubated at 37 °C for 24 h. Then, the cells were rinsed with PBS and fixed by 4% p-formaldehyde at room temperature for 30 min. Subsequent to the washing with PBS and staining for 5 min with 1 µM of 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Darmstadt, Germany), the cells were washed with PBS again and mounted. Fluorescence imaging was performed by using 470 nm excitation and 525 nm emission for GFP and 447 nm emission for DAPI with an EVOS digital inverted microscope (Life Technologies). Each experiment was done at least in triplicate and representative images were selected.

The method has been described [76 (link)]. Aliquots of 2 × 10⁴ U2OS-GFP-α-tubulin cells were seeded in each well of a sterile ibiTreat μ-slide (Ibidi, Germany), and cells were allowed to attach overnight. Cells were treated with 13.2 μg/mL Breastin or DMSO (solvent control) and incubated at 37 °C for 2 h. After rinsing with PBS and staining for 15 min with 300 nM 4′,6-diamidino-2-phenylindole (DAPI) (Life Technologies, Darmstadt, Germany), the cells were washed with PBS and mounted. Fluorescence imaging was performed by using 470 nm excitation and 525 nm emission for GFP and 360 nm excitation and 447 nm emission for DAPI with an EVOS digital inverted microscope (Life Technologies). Each experiment was repeated at least three times and representative images were selected.

Identification of myoblasts and myotubes was performed by immunofluorescence staining. Cells were stained with specific antibodies: either Pax7, Myf5, and MyoD for myoblast detection, or Myosin for myotube identification.
After two washing steps with PBS (Gibco^®Invitrogen™ Life Technologies, Carlsbad, CA, USA), cells were fixed and permeabilized with ice cold methanol for 20 min followed by several washing steps in PBS. Pax7 (ab199010, 1:200), Myf5 (M-18: sc-31949, 1:100), MyoD (M-318: sc-760, 1:150), and skeletal muscle Myosin (F59: sc-32732, 1:200, Santa Cruz Biotechnology, Dallas, TX, USA) were used as primary antibodies and incubated over night at 4 °C. After washing with 0.5% BSA/PBS cells were stained with Alexa Fluor^®488 (A11001 and A11008, 1:200, Invitrogen™ Life Technologies, Carlsbad, CA, USA), (Cy™3-conjugated donkey anti-Goat IgG (H+L) 705-165-003, 1:450, Jackson Immuno Research Laboratories, Inc, West Grove, PA, USA) (1h) in the dark at room temperature. After nuclei staining (15 min) with Hoechst 334,565, detection was performed using the EVOS^® Digital Inverted Microscope (Life Technologies, Carlsbad, CA, USA).
Experiments were performed 3 times using different myoblasts cells to confirm the results.

A dye exclusion test was used to determine the number of viable cells present in a cell suspension [85 ]. It is based on the principle that live cells possess intact cell membrane that exclude certain dyes such as trypan blue. In this test, a cell suspension is mixed with dye and then visually examined to determine, whether cells take up or exclude the dye. Viable cell have clear cytoplasms, whereas non-viable cells appear with blue cytoplasms. Briefly, 5 × 10⁵ cells were centrifuged, and the cell pellet was resuspended in 1 mL of medium. Twenty microliters of cell suspension were mixed with 20 μL of 0.4% trypan blue. Twenty microliters of the trypan blue/cell mixture were applied to a hemacytometer and cells were observed by EVOS digital inverted microscope (Life technologies GmbH, Darmstadt, Germany). The unstained (viable) and stained (nonviable) cells were counted and the percentage of viable cell was calculated as following.
Viable cells (%) = total number of viable cells per ml / total number of cells per mL × 100.