Mice were injected with 100 mg/kg ketamine and 20 mg/kg xylazine diluted in 0.9% isotone sodium chloride solution prior to intubation and surgery and kept narcotized using 1%–2% isoflurane in O2 for the time of imaging. Intravital imaging and surgery were carried out as described previously (24 (link), 52 (link)). After bone marrow surgery, the mouse was put into a 37°C warm NaCl bath, in which the bone marrow was imaged. Two-photon microscopy was done using a Leica TCS SP8 MP microscope with an HCX IRAPO L25×/0.95-NA water-immersion objective, 2 external hybrid reflected-light detectors (HyDs), and 2 external photomultiplier tubes (PMTs). Imaging was performed with a titanium-sapphire laser (Coherent Cameleon Vision II) tuned to 950 nm for intravital microscopy. FV-specific tdTomato+ CTLs (PMT, 585/40 filter), solid bone visualized by second-harmonic-generation (SHG) signal (HyD, 460/50 filter), CFSE-stained target cells (HyD, 525/50 filter), and QTracker 655–stained control cells (PMT 650/50 filter) or blood flow visualized through QTracker 655 (Invitrogen) were detected. For videos, one z-stack of 227.86 μm per minute or less time with a step size of 3 μm, an imaging speed of 400 Hz, and a pixel size of 1.16 was recorded in a 512 μm × 512 μm format for a collective video time of up to 3 hours. Videos were recorded using LAS X software (Leica Microsystems).
Qtracker 655
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Qtracker® 655 is a fluorescent quantum dot nanocrystal designed for biological labeling applications. It features a peak emission wavelength of 655 nanometers and can be used for various fluorescence-based techniques.
Automatically generated - may contain errors
Lab products found in correlation
R geco, by Addgene (2 mentions)
Ba460 500, by Olympus (2 mentions)
Ba520 560, by Olympus (2 mentions)
Fv1000mpe bx61wi upright microscope, by Olympus (2 mentions)
Insight deepsee ultrafast laser, by Spectra-Physics (2 mentions)
Xlpln25xwmp, by Olympus (2 mentions)
Ff01 425 30, by IDEX Corporation (2 mentions)
Las x software, by Leica (1 mentions)
Irdye 680 goat anti mouse igg, by LI COR (1 mentions)
Geltrex, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Roche (1 mentions)
Blasticidin s, by InvivoGen (1 mentions)
Irdye 800cw goat anti rabbit, by LI COR (1 mentions)
Pd0325901, by Fujifilm (1 mentions)
Puromycin, by InvivoGen (1 mentions)
Rhodamine dextran, by Thermo Fisher Scientific (1 mentions)
Qtracker 655 vascular label, by Thermo Fisher Scientific (1 mentions)
Rhodamine dextran, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Cell Signaling Technology (1 mentions)
Qtracker 585, by Thermo Fisher Scientific (1 mentions)
29 protocols using qtracker 655
1
Intravital Imaging of Bone Marrow
2
Immunoblotting Antibody Panel Protocol
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The following primary and secondary antibodies were used for immunoblotting: anti‐mouse OPN rabbit polyclonal antibody (IB1397; Immuno‐Biological Laboratories, Gunma, Japan), anti‐α‐tubulin (DM1A) mouse mAb (Millipore, Billerica, MA, USA), IRDye 680 goat anti‐mouse IgG and IRDye 800CW goat anti‐rabbit (LI‐COR Biosciences, Lincoln, NE, USA). Anti‐CD44 (GTX15834) was purchased from GeneTex (Irvine, CA, USA). Anti‐CD51 (104108), anti‐CD61 (104310), anti‐CD29 (102209), and rat or hamster IgG were purchased from BioLegend (San Diego, CA, USA). Phycoerythrin‐labeled anti‐Gr‐1 and allophycocyanin‐labeled anti‐Ly6G (1A8) were purchased from BioLegend. Collagen type C was purchased from Nitta Gelatin (Osaka, Japan). Blasticidin S and puromycin were purchased from InvivoGen (San Diego, CA, USA). Geltrex and Qtracker 655 were purchased from Thermo Fisher Scientific. DNase I was purchased from Roche Diagnostics (Basel, Switzerland). PD0325901 was purchased from Wako (Osaka, Japan).
3
Intravital Multiphoton Imaging of Bone Marrow
4
Cytotoxicity and Checkpoint Profiling of PBLs
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Activated PBLs were seeded at 1 × 106 cells per well in a U‐bottom 96‐well plate. Cancer cells were labelled with 10 nM Qtracker™ −655 (Thermo Fisher Scientific) according to manufacturer's instructions. A total of 2x103 cancer cells were added to the wells with activated PBLs (T:E ratio 1:50) and co‐cultured at 37°C, 5% CO2. After 4 h of direct co‐culture, the cells were harvested for interferon (IFN)‐γ ELISpot assay and cytotoxicity analysis by flow cytometry. In addition, 72 h direct co‐cultures of cancer cells and PBLs at T:E ratio of 1:20 were performed for immune checkpoint flow cytometry. The co‐culture supernatants were collected and stored at −80°C for Luminex array analysis.
5
Tracking Cell Fusion Dynamics
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Wild‐type and SMN‐deficient C2C12 cells were plated separately and differentiated for 2 days. Cells were then loaded with either QTracker 585 (Cat. #Q25011MP, Thermofisher) or QTracker 655 (Cat. #Q25021, Thermofisher) according to the manufacturer's instructions. Briefly, 106 cells were incubated with QTracker at 37°C and mixed every 15 min for 1 h. Cells were washed twice with differentiation media. Cells were mixed and re‐plated on coverslips and differentiated for an additional 5 days. Cells on coverslips were fixed with 4% paraformaldehyde and stained with DAPI (Cat. #4083S, Cell Signalling Technology, Danvers, MA, USA). Five images per condition were quantified for each n. Cell mixing images were quantified by dividing the number of double‐labelled cells by the total number of cells in the field of view.
6
In Vivo Imaging of Immune Cells
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Recombinant human C5a and anti-human C5aR mouse monoclonal IgG were purchased from EMD Millipore (Billerica, MA, USA) and Hycult Biotech (Uden, The Netherlands), respectively. A set of anti-Ki-67 antibody and EnVision+ solution was purchased from Dako; Agilent Technologies, Inc., (Santa Clara, CA, USA). Polyclonal anti-Ovalbumin mouse IgG and anti-BSA mouse IgG were purchased from Condrex (Redmond, WA, USA) and Rockland (Limerick, PA, USA), respectively. Ovalbumin and bovine serum albumin (BSA) were obtained from Sigma-Aldrich; Merck KGaA, (Darmstadt, Germany). Anti-mouse Ly6g rat IgG labeled with Cy-5® and anti-mouse CD11b rat IgG labeled with fluorescein isothiocyanate (FITC) were from Abcam (Cambridge, UK). The near-infrared ray cell labeling probes Qtracker 655 and Qtracker 800 were from Thermo Fisher Scientific, Inc., (Waltham, MA, USA). Other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan). Nude mice and Balb/c mice were supplied by Kyudo Experimental Animal Corp., (Kumamoto, Japan). The animal experiments were approved by the Kumamoto University Animal Experiment Committee (A 29–29) and performed according to the criteria of the Committee.
7
Intravital Microscopy of Murine Kidney
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Renal IVM was performed as previously described (27 (link)). Briefly, mice were anesthetized and tail veins catheterized for drug and reagent administration. The kidney was exteriorized using a lateral incision and extended over the heated imaging platform. Imaging was done with an SP5 Leica multiphoton confocal microscope (Leica, Germany) and MaiTai Ti-Sapphire laser (Spectra Physics, USA) at 800- to 850-nM excitation using predefined laser power and detector gain settings. Time-lapse imaging was acquired at 0.5 s per frame. Z stacks were acquired at 20-μm optical sections and 2-μm steps. The following reagents were administered intravenously to visualize physiological and cellular compartments: QTracker655 (Thermo Fisher Scientific, USA), phycoerythrin (PE)–conjugated anti-F4/80 (clone BM8, BioLegend, USA), PE-conjugated anti-CD11b (clone M1/70, BioLegend, USA), Alexa Fluor 568–conjugated LSALT peptide (conjugated using Protein Labeling Kit, Invitrogen, USA), and SYTOX Red (Thermo Fisher Scientific, USA). Native LASX software (Leica, Germany) was used for acquisition and image processing. Adherent leukocytes (LysM-GFP or antibody labeled) were manually counted in each field on the basis of their fixed position over the course of 2 min. A minimum of three fields were acquired and analyzed per mouse for quantification.
8
Vascular Leakage Analysis in Zebrafish
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Microangiography49 (link) was performed on SF, PA-CF, or cbs morphants mounted in 1.2% low-melt agarose prepared in zebrafish embryo medium50 . Embryos were injected with Qtracker 655 or 705 vascular labels (ThermoFisher Scientific) in the dorsal aorta around the yolk extension area using a pressure injector (World Precision Instruments, Friedberg, Germany). Injected embryos were immediately observed using an upright Leica TCS SP5 confocal microscope. Vascular leakages were analyzed in and around the head regions. At least three to five injected embryos were examined per dose in each experiment.
9
Isolation and Labeling of ECFCs
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Fresh human cord blood, donated under full ethical approval by healthy volunteers at the Northern Ireland Blood Transfusion Service (Belfast, U.K.), underwent density gradient fractionation for the isolation of mononuclear cells and was selected for ECFCs via resuspension in complete medium (EBM-2 plus EGM-2 MV supplements, Lonza Group) supplemented with 10% FBS and seeding onto 24-well culture plates precoated with rat tail collagen type 1 (BD Biosciences, Bedford, U.K.) at a density of 1 × 107 cells/mL. Cells were labeled (Qtracker 655, Invitrogen, Life Technologies, Carlsbad, CA) per the manufacturer’s instructions.
10
Intravitreal Injection of Stem Cells in OIR Mice
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