For imaging of unconjugated HEK cells to study ZAP70-mRuby2 recruitment to plasma membrane, cells were seeded directly into the imaging dish and transfected as described above. Prior to imaging, cells were washed gently in DPBS once and 1 ml of imaging medium was added to cover the cells in the dish. All live-cell imaging experiments in this study used a Nikon Ti inverted microscope in a thermally-controlled enclosure (OKOLabs), equipped with a CSU-X1 spinning-disc confocal head (Yokogawa). Laser lines used to excite samples were: 405 nm (100 mW), 488 nm (60 mW), 561 nm (50 mW) and 640 nm (100 mW), controlled by an acousto-optic tunable filter (Andor). Fluorescence emission was collected through filters for mTagBFP (460±15 nm), eGFP (525±25 nm), mRuby2 (607±18 nm) and IFP2.0 (708±38 nm). All images were collected using a Plan Apo VC 100× 1.4 NA oil-immersion objective (Nikon) onto an iXon Ultra EM-CCD camera (Andor) with a calculated pixel size of 85 nm. Stage movement was controlled by a Prior motorized stage with a Piezo Z-drive. The entire microscope system was controlled by μManager software that was used to create multi-channel, multi-time point image data-sets and at multiple positions when required. The built-in perfect-focus unit of the microscope was used to correct for axial focus drift due to fluctuations in temperature.
Csu x1 spinning disc confocal head
Manufactured by Yokogawa
Sourced in Japan
The CSU-X1 is a spinning disc confocal head developed by Yokogawa. It is designed to provide fast and high-resolution optical sectioning of samples for fluorescence microscopy applications. The core function of the CSU-X1 is to enable rapid image acquisition by using a rotating pinhole disc to simultaneously scan multiple points within the sample.
Automatically generated - may contain errors
Lab products found in correlation
Ixon ultra emccd camera, by Oxford Instruments (3 mentions)
Aotf controlled solid state lasers, by Oxford Instruments (3 mentions)
Ixon3 emccd camera, by Oxford Instruments (3 mentions)
Metamorph software, by Molecular Devices (3 mentions)
Ix81 inverted fluorescence microscope, by Olympus (3 mentions)
Plan apo vc 100 1.4 na oil immersion objective, by Nikon (2 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Mowiol, by Merck Group (1 mentions)
Dabco, by Merck Group (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Metamorph imaging software, by Universal Imaging (1 mentions)
Ti inverted microscope, by Nikon (1 mentions)
9 protocols using csu x1 spinning disc confocal head
1
Live-cell imaging of ZAP70-mRuby2 in HEK cells
2
Visualization of EEA1 and Lu/BCAM
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Cells were washed with phosphate buffered saline (PBS) and prefixed with ice cold methanol supplemented with 1 mM EGTA. After 10 min cells were transferred to 4% formaldehyde in PBS, washed, permeabilized with 0.15% Triton X-100 in PBS for 10 min and blocked by 1% BSA in PBS for 30 min. Incubation with the primary antibody (anti-EEA1 1∶200, Santa Cruz) (anti-Lu/BCAM 1∶150, Santa Cruz) was overnight at 4°C in PBS. Cells were washed with PBS and incubated with the suitable secondary antibody in PBS for 1 h at room temperature. Cells were washed, dried and embedded with Mowiol supplemented with DABCO (Sigma, St. Louis, MO, USA). Cells were analyzed with an inverted Axiovert 200 M microscope (Carl Zeiss GmbH, Jena, Germany), driven by Metamorph imaging software (Universal Imaging, Downingtown, PA, USA), with a Yokogawa CSU-X1 spinning disc confocal head (Tokyo, Japan) with emission filter wheel, with a Coolsnap HQ II digital camera and with 488 nm and 561 nm laser lines. Images were processed with Metamorph software.
3
Immunofluorescence Staining Protocol
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Cells were fixed for 15 min with 4% formaldehyde in PBS, permeabilized for 5 min with 0.1% TritonX-100 in PBS and then blocked with 2% BSA in PBS for 1 hour. Cells were stained with primary antibodies at 1:200 dilution in blocking buffer and, after further washing, with secondary antibody (anti-rabbit AlexaFluor 647, 1:500 dilution) in blocking buffer for 1 h. Samples were washed thoroughly and cover slips mounted on microscopy glasses with ProLong Gold anti-fade reagent with DAPI (Invitrogen). Imaging was performed using Nikon Ti microscope equipped with CSU-X1 spinning disc confocal head (Yokogawa) and with Zeiss 780 system.
4
Live-Cell Imaging of Transfected Cells
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For routine live-cell imaging of transfected cells, we used a Nikon Ti inverted microscope equipped with a CSU-X1 spinning-disc confocal head (Yokogawa) and maintained at 37°C/5% CO2 with a thermally controlled enclosure (OKOLabs). Unless otherwise specified, a Plan Apo VC 100×/NA 1.4 oil-immersed objective (Nikon) was used for imaging onto an iXon Ultra EM-CCD camera (Andor) with a calculated pixel size of 134 nm, with 405, 488, 561, and 640 nm laser lines for fluorescent excitation. Fluorescence emission was collected through filters for mTagBFP (460 ± 15 nm), mGFP (525 ± 25 nm), mCherry/mScarlet (607 ± 18 nm), AF647/APC (685 ± 20 nm), and iRFP713 (708 ± 38 nm). The entire microscope system was controlled by μManager2 software that was used to create multichannel, multitime-point image data sets, and at multiple positions when required. The built-in perfect-focus unit of the microscope was used to correct axial focus drift due to fluctuations in temperature.
5
Live-cell imaging of ZAP70-mRuby2 in HEK cells
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For imaging of unconjugated HEK cells to study ZAP70-mRuby2 recruitment to plasma membrane, cells were seeded directly into the imaging dish and transfected as described above. Prior to imaging, cells were washed gently in DPBS once and 1 ml of imaging medium was added to cover the cells in the dish. All live-cell imaging experiments in this study used a Nikon Ti inverted microscope in a thermally-controlled enclosure (OKOLabs), equipped with a CSU-X1 spinning-disc confocal head (Yokogawa). Laser lines used to excite samples were: 405 nm (100 mW), 488 nm (60 mW), 561 nm (50 mW) and 640 nm (100 mW), controlled by an acousto-optic tunable filter (Andor). Fluorescence emission was collected through filters for mTagBFP (460±15 nm), eGFP (525±25 nm), mRuby2 (607±18 nm) and IFP2.0 (708±38 nm). All images were collected using a Plan Apo VC 100× 1.4 NA oil-immersion objective (Nikon) onto an iXon Ultra EM-CCD camera (Andor) with a calculated pixel size of 85 nm. Stage movement was controlled by a Prior motorized stage with a Piezo Z-drive. The entire microscope system was controlled by μManager software that was used to create multi-channel, multi-time point image data-sets and at multiple positions when required. The built-in perfect-focus unit of the microscope was used to correct for axial focus drift due to fluctuations in temperature.
6
Immunofluorescence Staining of Periphilin
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Cells were grown on glass cover slips and then fixed with 4% formaldehyde in PBS for 15 min. Cells were permeabilized with 0.1% Triton X100 in PBS and then blocked with 5% BSA in PBS. Samples were stained with primary anti-Periphilin antibody (Atlas, HPA038902) at dilution 1:500 for 1 h and after washing with blocking buffer with secondary anti-rabbit AlexaFluor 568 antibody diluted 1/500 for 1 h. Cover slips were mounted on microscopy glasses with ProLong Gold anti-fade reagent with DAPI (Invitrogen). Imaging was performed using Nikon Ti microscope equipped with CSU-X1 spinning disc confocal head (Yokogawa) and with Zeiss 780 system.
7
Peptide Vesicle Imaging Techniques
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All the peptide vesicles images were acquired using an oil immersion 60×/1.4 NA Plan-Apochromat objective lens mounted on an Olympus IX-81 inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with a CSU-X1 spinning disc confocal head (Yokogawa Electric Corporation, Tokyo, Japan), AOTF-controlled solid-state lasers (Andor Technology, Belfast, UK), and an iXON3 EMCCD camera (Andor Technology, Belfast, UK).
Fluorescence images were captured using MetaMorph software (Molecular Devices, San Jose, CA) with laser excitation at 488 nm for GFP, DiO, and Green Lys and 561 nm for Rho-PE and rhodamine. Image analysis was performed using NIH-ImageJ.
Fluorescence images were captured using MetaMorph software (Molecular Devices, San Jose, CA) with laser excitation at 488 nm for GFP, DiO, and Green Lys and 561 nm for Rho-PE and rhodamine. Image analysis was performed using NIH-ImageJ.
8
Peptide Vesicle Imaging Workflow
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All the peptide vesicles images were acquired using an oil immersion 60×/1.4 NA Plan-Apochromat objective lens mounted on an Olympus IX-81 inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with a CSU-X1 spinning disc confocal head (Yokogawa Electric Corporation, Tokyo, Japan), AOTF-controlled solidstate lasers (Andor Technology, Belfast, UK), and an iXON3 EMCCD camera (Andor Technology, Belfast, UK). Fluorescence images were captured using MetaMorph software (Molecular Devices, San Jose, CA) with laser excitation at 488 nm for GFP, DiO, and Green Lys and 561 nm for Rho-PE and rhodamine. All the image analysis was performed using NIH-ImageJ.
9
Peptide Vesicle Imaging Techniques
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All the peptide vesicles images were acquired using an oil immersion 60×/1.4 NA Plan-Apochromat objective lens mounted on an Olympus IX-81 inverted fluorescence microscope (Olympus Corporation, Tokyo, Japan) equipped with a CSU-X1 spinning disc confocal head (Yokogawa Electric Corporation, Tokyo, Japan), AOTF-controlled solid-state lasers (Andor Technology, Belfast, UK), and an iXON3 EMCCD camera (Andor Technology, Belfast, UK).
Fluorescence images were captured using MetaMorph software (Molecular Devices, San Jose, CA) with laser excitation at 488 nm for GFP, DiO, and Green Lys and 561 nm for Rho-PE and rhodamine. Image analysis was performed using NIH-ImageJ.
Fluorescence images were captured using MetaMorph software (Molecular Devices, San Jose, CA) with laser excitation at 488 nm for GFP, DiO, and Green Lys and 561 nm for Rho-PE and rhodamine. Image analysis was performed using NIH-ImageJ.
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