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 brightfield and fluorescent images of GFP + /tdTomato + double‐positive AEC‐tLgT cells after FACS sorting and expansion. Images were taken at 10× magnification on an ECHO Revolve R4 fluorescence microscope. Scale bar, 100 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: 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

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 images of AEC‐tLgT organoids after 2 months of growth. Large panel is a brightfield image of one Transwell taken at 4× magnification. Scale bar, 900 µm. Small panels were taken under both brightfield and fluorescence at 10× magnification. Scale bar, 100 µm. Images were taken 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

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 data and images for CSC‐ and NiCl 2 ‐treated AEC‐tLgT cells. ( A ) RT‐qPCR was used to measure induction of xenobiotic response‐related genes CYP1A1 and CYP1B1 after 48 hr CSC treatment of AEC‐tLgT cells. mRNA abundance was normalized to actin ( ACTB ) levels and fold change was calculated over DMSO vehicle‐treated samples. N = 3. Values shown as mean ± SD . ( B ) Representative brightfield images at 4× magnification of vehicle‐ and CSC‐treated samples showing progressive morphological changes with increasing CSC dose. ( C ) Representative RT‐qPCR results for vehicle‐ and nickel (NiCl 2 )‐treated AEC‐tLgT cells after 48 hr showing induction of metal response genes CA9 and NDRG1 . mRNA abundance was normalized to MTF1 levels and fold change was calculated over water vehicle‐treated samples. N = 3. Values shown as mean ± SD . ( D ) Representative brightfield images at 4× magnification of vehicle‐ and NiCl 2 ‐treated samples showing stable cell survival across NiCl 2 .

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: Quantitative RT-PCR, Stable Transfection