Journal: MethodsX

Article Title: Production of isoform-specific knockdown/knockout Madin–Darby canine kidney epithelial cells using CRISPR/Cas9

doi: 10.1016/j.mex.2020.101149

Figure Lengend Snippet: pspCas9(BB)−2A-GFP plasmids transfection into MDCK cells. Fluorescence microscopy depicting brightfield (top) and corresponding GFP (bottom) images of MDCK cells transfected with pspCas9(BB)−2A-GFP plasmids encoding sgRNAs that A) recognize intronic targets upstream and B) downstream of CXADR exon 8. Images were taken on an Olympus IX83 microscope with an Olympus DP74 microscope digital camera using Olympus cellSens software. Images were taken at 10X magnification. Brightness and contrast of images were adjusted using Photoshop software. Black bars represent 50 µm.

Article Snippet: • Biosafety cabinet • 37 °C incubated orbital shaker • 37 °C humidified incubator with 5% CO 2 • Micropipettors and tips • Multichannel micropipettor • Pipet aid with 5, 10, and 25 ml serological pipettes • 500 mL capacity 0.2 μm filter units • 0.22 μm syringe filters • 1.5 ml microfuge tubes • Table top microtube centrifuge, 4 °C and room temperature (RT) • 50 °C heating block • Nanodrop 2000 (ThermoFisher, Waltham MA) • Olympus IX83 Microscope (Olympus Corporation, Tokyo, Japan) • Olympus DP74 Microscope Digital Camera (Olympus Corporation, Tokyo, Japan) • Olympus cellSens software (Olympus Corporation, Tokyo, Japan) • Countess II FL Automated Cell Counter (Thermofisher, Waltham, MA) • Nunc Lab-Tek 4-well Chamber Slide with removable wells (Cat# 154,526; ThemoFisher, Waltham, MA) • Cytoone 24-well plates (Cat# CC7682–7524; USA Scientific, Ocala, FL) • Photoshop Software • Becton Dickinson FACSAria III system (Franklin Lakes, NJ) • Computer and internet access • Mastercycler nexus eco (Eppendorf, Hamburg, Germany) • Milli Q water purification system (EMD Millipore, Burlington, MA)

Techniques: Transfection, Fluorescence, Microscopy, Software

Journal: MethodsX

Article Title: Production of isoform-specific knockdown/knockout Madin–Darby canine kidney epithelial cells using CRISPR/Cas9

doi: 10.1016/j.mex.2020.101149

Figure Lengend Snippet: pspCas9(BB)−2A-GFP plasmids transfection into MDCK cells. Fluorescence microscopy depicting brightfield (top) and corresponding GFP (bottom) images of MDCK cells transfected with pspCas9(BB)−2A-GFP plasmids encoding sgRNAs that A) recognize intronic targets upstream and B) downstream of CXADR exon 8. Images were taken on an Olympus IX83 microscope with an Olympus DP74 microscope digital camera using Olympus cellSens software. Images were taken at 10X magnification. Brightness and contrast of images were adjusted using Photoshop software. Black bars represent 50 µm.

Article Snippet: • Biosafety cabinet • 37 °C incubated orbital shaker • 37 °C humidified incubator with 5% CO 2 • Micropipettors and tips • Multichannel micropipettor • Pipet aid with 5, 10, and 25 ml serological pipettes • 500 mL capacity 0.2 μm filter units • 0.22 μm syringe filters • 1.5 ml microfuge tubes • Table top microtube centrifuge, 4 °C and room temperature (RT) • 50 °C heating block • Nanodrop 2000 (ThermoFisher, Waltham MA) • Olympus IX83 Microscope (Olympus Corporation, Tokyo, Japan) • Olympus DP74 Microscope Digital Camera (Olympus Corporation, Tokyo, Japan) • Olympus cellSens software (Olympus Corporation, Tokyo, Japan) • Countess II FL Automated Cell Counter (Thermofisher, Waltham, MA) • Nunc Lab-Tek 4-well Chamber Slide with removable wells (Cat# 154,526; ThemoFisher, Waltham, MA) • Cytoone 24-well plates (Cat# CC7682–7524; USA Scientific, Ocala, FL) • Photoshop Software • Becton Dickinson FACSAria III system (Franklin Lakes, NJ) • Computer and internet access • Mastercycler nexus eco (Eppendorf, Hamburg, Germany) • Milli Q water purification system (EMD Millipore, Burlington, MA)

Techniques: Transfection, Fluorescence, Microscopy, Software

Journal: MethodsX

Article Title: Production of isoform-specific knockdown/knockout Madin–Darby canine kidney epithelial cells using CRISPR/Cas9

doi: 10.1016/j.mex.2020.101149

Figure Lengend Snippet: Parental MDCK cells and JR1-CAR Ex8 -KO MDCK exhibit similar morphology. Parental MDCK cells (left) and JR1-CAR Ex8 -KO cells (right) were seeded on chamber slides at similar concentrations. Bright field microscopy images were taken at 24, 48, and 72 h post seeding. Images were taken on an Olympus IX83 microscope with an Olympus DP74 microscope digital camera using Olympus cellSens software. Images were taken at 10X magnification. Black bars represent 200 µm.

Article Snippet: • Biosafety cabinet • 37 °C incubated orbital shaker • 37 °C humidified incubator with 5% CO 2 • Micropipettors and tips • Multichannel micropipettor • Pipet aid with 5, 10, and 25 ml serological pipettes • 500 mL capacity 0.2 μm filter units • 0.22 μm syringe filters • 1.5 ml microfuge tubes • Table top microtube centrifuge, 4 °C and room temperature (RT) • 50 °C heating block • Nanodrop 2000 (ThermoFisher, Waltham MA) • Olympus IX83 Microscope (Olympus Corporation, Tokyo, Japan) • Olympus DP74 Microscope Digital Camera (Olympus Corporation, Tokyo, Japan) • Olympus cellSens software (Olympus Corporation, Tokyo, Japan) • Countess II FL Automated Cell Counter (Thermofisher, Waltham, MA) • Nunc Lab-Tek 4-well Chamber Slide with removable wells (Cat# 154,526; ThemoFisher, Waltham, MA) • Cytoone 24-well plates (Cat# CC7682–7524; USA Scientific, Ocala, FL) • Photoshop Software • Becton Dickinson FACSAria III system (Franklin Lakes, NJ) • Computer and internet access • Mastercycler nexus eco (Eppendorf, Hamburg, Germany) • Milli Q water purification system (EMD Millipore, Burlington, MA)

Techniques: Microscopy, Software