mlg mouse lung fibroblasts  (ATCC)

Journal: Current Protocols

Article Title: Immortalization of Human Adult Alveolar Epithelial Cells to Study Environmental Exposures of the Distal Lung

doi: 10.1002/cpz1.70372

Figure Lengend Snippet: Schematic illustration of 3D co‐culture setup using AEC‐tLgT epithelial cells and MLg mouse fibroblasts. Epithelial cells and fibroblasts are mixed with Matrigel and loaded onto Transwell filters and provided nutrients through medium perfusion from the outside chamber. GFR, growth factor‐reduced; PET, polyester.

Article Snippet: MLg mouse lung fibroblasts (ATCC, CCL‐206, RRID:CVCL_0437) MLF medium (MLg fibroblast culture medium, see recipe) Fmed+ROCKinh culture medium (see recipe) Sterile 1× DPBS (Corning, 21‐031‐CV) 0.05% (w/v) trypsin‐EDTA solution (Gibco, 25300‐062) 0.4% trypan blue staining (Gibco, 15250‐061) AEC‐tLgT immortalized cells (from Basic Protocol ) GFR Matrigel (Corning, 354230) 3D basic medium (see recipe) Accutase (Innovative Cell Technologies, AT‐104) SB‐431542, working stock of 1 mM (Selleck Chemicals, S1067) 15‐ and 50‐ml conical tubes Benchtop centrifuge that can accommodate 15‐ and 50‐ml conical tubes Hemocytometer (Thermo Fisher Scientific, 02‐671‐5) 15‐cm tissue culture dish (Corning, CLS430599) or T‐175 culture flask with vented cap T‐25 culture flask with vented cap 8‐ or 10‐in. length straight specimen forceps, serrated (e.g., VWR, 82027‐436 or ‐434) 24‐well Transwell inserts (Corning, 3470) 24‐well tissue culture plate 1.5‐ml microcentrifuge tubes Bucket or container of ice to bring into the tissue culture hood Sealed 200‐μl pipette filter tips, pre‐cooled at 4°C to be used in the hood (optional) Brightfield microscope with either 4× or 2.5× objective

Techniques: Co-Culture Assay

Journal: Current Protocols

Article Title: Immortalization of Human Adult Alveolar Epithelial Cells to Study Environmental Exposures of the Distal Lung

doi: 10.1002/cpz1.70372

Figure Lengend Snippet: Representative cytospin immunofluorescence staining results to assess the purity of isolated AT2 cells. This example was from a lung preparation yielding 93% viable EpCAM + cells and 91% AT2 cell purity. CD45, leukocyte cell marker; VIM, vimentin fibroblast marker; CC10, Club cell marker; TUBB4A, ciliated cell marker (airway); KRT5, basal cell marker (airway); MUC5B, goblet cell marker (airway); NKX2‐1, general lung cell marker; HTII‐280 and SPC, AT2 cell markers; mouse IgG, rabbit IgG, and mouse IgM are negative controls. Cytocentrifuged cells were nuclear counterstained with propidium iodide (red). Images were taken at 10× magnification on a Nikon Ti Eclipse fluorescence microscope. Scale bar, 50 µm.

Article Snippet: MLg mouse lung fibroblasts (ATCC, CCL‐206, RRID:CVCL_0437) MLF medium (MLg fibroblast culture medium, see recipe) Fmed+ROCKinh culture medium (see recipe) Sterile 1× DPBS (Corning, 21‐031‐CV) 0.05% (w/v) trypsin‐EDTA solution (Gibco, 25300‐062) 0.4% trypan blue staining (Gibco, 15250‐061) AEC‐tLgT immortalized cells (from Basic Protocol ) GFR Matrigel (Corning, 354230) 3D basic medium (see recipe) Accutase (Innovative Cell Technologies, AT‐104) SB‐431542, working stock of 1 mM (Selleck Chemicals, S1067) 15‐ and 50‐ml conical tubes Benchtop centrifuge that can accommodate 15‐ and 50‐ml conical tubes Hemocytometer (Thermo Fisher Scientific, 02‐671‐5) 15‐cm tissue culture dish (Corning, CLS430599) or T‐175 culture flask with vented cap T‐25 culture flask with vented cap 8‐ or 10‐in. length straight specimen forceps, serrated (e.g., VWR, 82027‐436 or ‐434) 24‐well Transwell inserts (Corning, 3470) 24‐well tissue culture plate 1.5‐ml microcentrifuge tubes Bucket or container of ice to bring into the tissue culture hood Sealed 200‐μl pipette filter tips, pre‐cooled at 4°C to be used in the hood (optional) Brightfield microscope with either 4× or 2.5× objective

Techniques: Immunofluorescence, Staining, Isolation, Marker, Fluorescence, Microscopy

Journal: Current Protocols

Article Title: Immortalization of Human Adult Alveolar Epithelial Cells to Study Environmental Exposures of the Distal Lung

doi: 10.1002/cpz1.70372

Figure Lengend Snippet: Representative brightfield images of MLg fibroblasts at 50% confluence, the optimal growth pattern to set up 3D co‐cultures with AEC‐tLgT cells. Images taken at 4× magnification (left; scale bar, 900 µm) and 10× magnification (right; scale bar, 360 µm) on an ECHO Revolve R4 fluorescence microscope.

Article Snippet: MLg mouse lung fibroblasts (ATCC, CCL‐206, RRID:CVCL_0437) MLF medium (MLg fibroblast culture medium, see recipe) Fmed+ROCKinh culture medium (see recipe) Sterile 1× DPBS (Corning, 21‐031‐CV) 0.05% (w/v) trypsin‐EDTA solution (Gibco, 25300‐062) 0.4% trypan blue staining (Gibco, 15250‐061) AEC‐tLgT immortalized cells (from Basic Protocol ) GFR Matrigel (Corning, 354230) 3D basic medium (see recipe) Accutase (Innovative Cell Technologies, AT‐104) SB‐431542, working stock of 1 mM (Selleck Chemicals, S1067) 15‐ and 50‐ml conical tubes Benchtop centrifuge that can accommodate 15‐ and 50‐ml conical tubes Hemocytometer (Thermo Fisher Scientific, 02‐671‐5) 15‐cm tissue culture dish (Corning, CLS430599) or T‐175 culture flask with vented cap T‐25 culture flask with vented cap 8‐ or 10‐in. length straight specimen forceps, serrated (e.g., VWR, 82027‐436 or ‐434) 24‐well Transwell inserts (Corning, 3470) 24‐well tissue culture plate 1.5‐ml microcentrifuge tubes Bucket or container of ice to bring into the tissue culture hood Sealed 200‐μl pipette filter tips, pre‐cooled at 4°C to be used in the hood (optional) Brightfield microscope with either 4× or 2.5× objective

Techniques: Fluorescence, Microscopy