A PDMS microfluidics slide chip (Abnova) was coated with 1 mg/mL streptavidin (Agilent). The chip was then washed and coated with 0.4 mg/mL biotin-vimentin (MD Anderson) for 1 h. Next, 1.5 × 106 attIL12-PBMCs (labeled with Calcein Green AM [ThermoFisher]) and 1.5 × 106 control-PBMCs (labeled with CMTPX red cell tracker [ThermoFisher]) were mixed at a ratio of 1:1, loaded into a spiral chamber (Abnova), and passed through the slide chip at a flow rate of 1.8 mL per hour using the CytoQuest microfluidics pump (Abnova). The slide chip was then imaged on a fluorescent microscope (Keyence). Cells were counted using Keyence BZ-X700 analysis software.
Cmtpx red cell tracker
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
The CMTPX Red Cell Tracker is a fluorescent dye that can be used to label and track live cells in vitro. It provides a bright, stable red fluorescent signal that is well-retained within the cell membrane, allowing for long-term monitoring of cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Calcein green am, by Thermo Fisher Scientific (2 mentions)
Fluorescent microscope, by Keyence (2 mentions)
Spiral chamber, by Abnova (2 mentions)
Bz x700 analysis software, by Keyence (2 mentions)
Celltracker green cmfda, by Thermo Fisher Scientific (1 mentions)
Glass bottom petri dish, by Ibidi (1 mentions)
Widefield system, by Nikon (1 mentions)
4 protocols using cmtpx red cell tracker
1
Microfluidic Assay for Immune Cell Binding
2
Vimentin-Specific T Cell Capture
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A polydimethylsiloxane microfluidics slide chip (Abnova) was coated with 1 mg/mL streptavidin (Agilent). The chip was then washed and coated with 0.4 mg/mL biotin-vimentin (MD Anderson) for 1 hour. Next, 1.5×106 attIL12-T cells (labeled with Calcein Green AM; ThermoFisher) and 1.5×106 control-T cells (labeled with CMTPX red cell tracker; ThermoFisher) were mixed at a ratio of 1:1, loaded into a spiral chamber (Abnova), and passed through the slide chip at a flow rate of 1.8 mL per hour using the Cytoquest microfluidics pump (Abnova). The slide chip was then imaged on a fluorescent microscope (Keyence). Cells were enumerated using Keyence BZ-X700 analysis software.
3
Live Cell Fluorescent Imaging
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The cells cultured on free‐standing substrate were labelled with Cell TrackerTM Red CMTPX (C34552, Invitrogen, Loughborough, UK) for live cell fluorescent and confocal microscopy during cell culture. An aliquot of 5 μL fluorescent probe Cell TrackerTM reagent stock solution prepared in DMSO was directly added into the cell culture medium to the working concentration (0.5–25 μM). After incubated for 24 h at 37°C and 5% CO2, the medium with Cell TrackerTM reagent was replenished with fresh medium. Cell culture was then continued and the labelled cells were imaged and analysed using fluorescent (Nikon Ti) and confocal microscopes (Nikon Cl, Japan) at λex = 580 nm, λem = 650 nm (for TRITC/Cell TrackerTM visualisation).
4
Live-cell Imaging of Myoblast Co-culture
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Live cell images were acquired using the inverted Ti eclipse Nikon Widefield system equipped with a Plan Apo 20× (NA 0.75) objective, SpectraX LED excitation (395 nm, 440 nm, 470 nm, 508 nm, 561 nm, 640 nm), Quad filter for DAPI/GFP/RFP/Cy5, and Andor Zyla sCMOS camera (2560 × 2160; 6.5 μm pixels). The environmental conditions of 37 °C with 5% CO2 were maintained using the Oko-lab environmental control chamber. Image acquisition used the NIS Elements Software Version 5.21.00 .
Control and DAG1 KO myoblasts were stained with CellTrackerTM Red CMTPX (Invitrogen, Waltham, MA, USA) and CellTrackerTM Green CMFDA (Invitrogen, Waltham, MA, USA), respectively. Control and KO cells were then co-cultured in a glass bottom petri dish (Ibidi, Fitchburg, WI, USA). Each experiment was imaged over 15 h with each XY position imaged every 10 min. For each time point, a brightfield image was taken in addition to images in the red and green channels to identify the two cell types.
Control and DAG1 KO myoblasts were stained with CellTrackerTM Red CMTPX (Invitrogen, Waltham, MA, USA) and CellTrackerTM Green CMFDA (Invitrogen, Waltham, MA, USA), respectively. Control and KO cells were then co-cultured in a glass bottom petri dish (Ibidi, Fitchburg, WI, USA). Each experiment was imaged over 15 h with each XY position imaged every 10 min. For each time point, a brightfield image was taken in addition to images in the red and green channels to identify the two cell types.
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