Human dermal fibroblasts were isolated from the foreskin of a 5 y. o. male as described before68 . The tissue was washed 3 times in PBS (Gibco, Carlsbad, CA, USA) and digested on the dermal side in 2.8 ml/60 cm2 dispase type II (Roche, Basel, Switzerland) and incubated for 20 h at 4 °C. The epidermis and dermis was separated using a forceps, the dermal tissue was placed in a tissue culture plate and 1.5 ml FGM (Gibco, Carlsbad, CA, USA) was added. The enzymatic reaction was stopped by Trypsin (all Sigma-Aldrich, St. Louis, MO, USA) with 10 ml FGM consisting of DMEM medium with 10% FCS, 1% glutamine and 1% Pen/Strep (all Gibco, Carlsbad, CA, USA). The cells’ suspension was filtered using a cell strainer and washed with 5 ml PBS. Cells were centrifuged at 130 g for 5 min at 25 °C, the supernatant discarded and suspended in 10 ml PBS. The medium was changed every other day and cultured for 4 days at 37 °C. The purity was regularly controlled optically and reached approx. 90%. The cells were suspended in a collagen matrix NMSC model on the 18 mm diameter microscope cover glass (VWR, Darmstadt, Germany). The fibroblasts were cultured for 26 h at 37 °C before the TPE-FLIM measurements.
Microscope cover glass
Manufactured by Avantor
Sourced in Germany
Microscope cover glass is a thin, transparent glass sheet used to cover specimens placed on a microscope slide. Its primary function is to protect the specimen and provide a flat, uniform surface for optimal imaging quality.
Automatically generated - may contain errors
Lab products found in correlation
Pen strep, by Thermo Fisher Scientific (1 mentions)
Dispase type 2, by Roche (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
Interleukin 4 (il 4), by Thermo Fisher Scientific (1 mentions)
Gm csf, by Thermo Fisher Scientific (1 mentions)
Tryptan blue, by Merck Group (1 mentions)
Human serum albumin (hsa), by Thermo Fisher Scientific (1 mentions)
Centrifuge 5415r, by Eppendorf (1 mentions)
Cell strainer, by Thermo Fisher Scientific (1 mentions)
Bright line hemocytometer, by Merck Group (1 mentions)
Accumet ae150, by Thermo Fisher Scientific (1 mentions)
Ckx41, by Olympus (1 mentions)
3 protocols using microscope cover glass
1
Isolation and Culture of Human Dermal Fibroblasts
2
Dendritic Cell Differentiation from CD14+ PBMCs
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CD14 positive PBMCs were used to differentiate dendritic cells by washing in PBS and centrifuging at 350×g for 10 min two times. About 5 ml RPMI medium, supplemented with 1% Pen/Strep and 1% FCS (Biochtrom), was added. Tryptan Blue (Sigma-Aldrich) was used for counting the cells in a hemocytometer, seeded at 2.0×106 cells/ml and incubated for 2 hr at 37°C under 5% CO2. Non-attached cells and the supernatant were discarded. Adding 500 µl basal Iscove’s medium to the cells supplemented with 1% Pen/Strep, 1% glutamine, 5% HSA (all Gibco), 100 ng/ml IL-4, 100 ng/ml GM-CSF (both Peprotech) with medium change every second day for 6 days at 37°C. For TPE-FLIM imaging, the cells were seeded on 18 mm diameter microscope cover glass (VWR) in PBS containing Ca2+ at room temperature.
3
Protoplast Isolation and Characterization
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Single edge industrial razor blades (VWR, Missouri, TX, USA, Cat no. 55411-050).
Fluorescence microscope (Olympus CKX41, Olympus, Center Valley, PA, USA).
Confocal laser scanning microscope (Leica SM8, Leica, Wetzlar, Germany).
Bench Centrifuge with swinging-bucket rotor (Sovrall Legend RT, Kendro Laboratory Products, Newtown, CT, USA).
Centrifuge 5415 R (Eppendorf, Hamburg, Germany).
Orbital Incubator shaker for bacterial growth (GyroMax 737, Amerex Instruments Inc, Concord, CA, USA).
Orbital shaker for protoplast digestion (New Brunswick Scientific, Edison, NJ).
pH meter (Accumet AE150, Fisher Scientific).
14-ml polystyrene round-bottomed tube (Becton, Dickinson Labware, NJ, USA).
Cell Strainer, (70 µm nylon mesh, Fisher Scientific, Cat. no. 22363548,).
12-well Falcon Plates (Cat. no. 351143, Corning Incorporated, Corning, NY. USA).
Petri dishes (60 × 15 mm, cat. no. 430196, Corning Incorporated).
Glass bottom dish (35 mm dish with 14 mm bottom well, In Vitro Scientific, Cat. no. D35-14-1-N).
Bright-Line Hemocytometer (Sigma-Aldrich, Cat. no. Z359629).
Microscope cover glass (VWR, Cat. no. 48366067).
Vacuum desiccator (Bel-Art Products, Pequannock, NJ, USA, Cat. no. F42027).
Vacuum pump (Welch vacuum, Thomas Industries Inc., Skokie, IL, USA).
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