The following reagents were used: BSA (PAA Laboratories), hexadecyltrimethylammonium bromide (HTAB; Acros Organics), isoflurane (Forene 100%; Abbott), and recombinant mouse complement component 5a (C5a; R&D Systems). Calcein green, calcein red-orange, fluo-3 AM, FluoSpheres polystyrene microbeads orange (1 µm), and PTX were obtained from Life Technologies. LPS (from Pseudomonas aeruginosa), Evans blue, histamine, and tetramethylbenzidine (TMB) substrate were purchased from Sigma-Aldrich.
Calcein red orange
Manufactured by Thermo Fisher Scientific
Sourced in United States
Calcein Red-Orange is a fluorescent dye used for labeling and tracking cells. It emits an orange-red fluorescence when excited by a suitable light source. The dye can be used to stain live cells and monitor their viability, proliferation, and other cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Calcein green, by Thermo Fisher Scientific (4 mentions)
Calcein am, by Thermo Fisher Scientific (3 mentions)
Calcein, by Thermo Fisher Scientific (2 mentions)
Celltrace, by Thermo Fisher Scientific (2 mentions)
Histamine, by Merck Group (1 mentions)
Forene 100, by Abbott (1 mentions)
Tetramethylbenzidine tmb substrate, by Merck Group (1 mentions)
Fluo 3 am, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by GE Healthcare (1 mentions)
Er tracker red, by Thermo Fisher Scientific (1 mentions)
Tubulin tracker green, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Environmental chamber, by Okolab (1 mentions)
Volocity 3d image analysis software, by PerkinElmer (1 mentions)
Fluorescein isothiocyanate (fitc), by Merck Group (1 mentions)
Nis element, by Nikon (1 mentions)
Elyra ps 1 lsm 780, by Zeiss (1 mentions)
Zen 2011, by Zeiss (1 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (1 mentions)
Penicillin streptomycin, by Fujifilm (1 mentions)
27 protocols using calcein red orange
1
Murine Inflammatory Assays
2
Multimodal Imaging of Cell Membranes and Organelles
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Stock solutions of Ap3 and FM4-64 were diluted in the imaging buffer (125 mM NaCl, 5 mM KCl, 10 mM dextrose, 10 mM HEPES, 1 mM MgCl2 and 2 mM CaCl2, pH 7.3) and applied to the cells at a final concentration of 10–20 μM. Both dyes were readily adsorbed to the membrane and reached equilibrium within minutes, without any obvious cellular toxicity during the imaging period (Supplementary Fig. 5 ). For multimodal imaging experiments, pHrodo Green Dextran (Life Technologies, 20 μg ml−1), Calcein Red-Orange (Life Technologies, 0.5 μM), Tubulin Tracker Green (Life Technologies, 0.5 μM), ER-Tracker Red (Life Technologies, 5 μM) and Calcein (Life Technologies, 0.5 μM) were applied to cells in the imaging buffer at the indicated concentration and incubated at 37 °C for 30 min–1 h before imaging. For multimodal imaging with EGFP and DsRed, a total of 1.5–2.0 μg of pEGFP-N1 and pDsRed-N1 empty vectors was transfected into CHO cells using 3.0–4.0 μl of Lipofectamine 2000 (Life Technologies) following the manufacturer's protocol, and imaging was performed 24–60 h after transfection.
3
Visualizing Bacterial Internalization in MG-63 Cells
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To confirm the bacterial internalization, MG-63 cells were seeded onto slides after the internalization assay and then Gram and Giemsa stained. For live confocal microscopy, MG-63 cells were seeded onto Ibidi® μ-Slide 18 well (Munich, Germany) and infected with two selected clinical strains (ATCC6919 and BL), as described above. Fluorescein isothiocyanate (Sigma-Aldrich®, Saint Quentin Fallavier, France) was used to visualize the bacteria, along with calcein red/orange (Thermo Fischer Scientific, Waltham, MA USA) to label the cellular membrane. Slides were acquired on a Nikon A1 Rsi confocal microscope designed for live cell imaging using an Okolab environmental chamber to regulate temperature and air/CO2/N2. Pictures were analyzed with Fiji software29 (link). 3D images were processed with NIS elements (Nikon Instruments Inc.) and Volocity 3D Image Analysis Software (PerkinElmer).
4
Visualizing Primary NK Cell Migration
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First, primary NK cells were stained with 1 µM Calcein Red-Orange (Thermo Fisher Scientific), and then labeled cells were incubated at 5 × 105 cells/ml into coated chambered glass coverslips in clone media. Where indicated, 1 µM TAPI-0 was added to wells before imaging. Time-lapse imaging was performed at 37°C and 5% CO2 immediately after cells landed on slides at rate of one frame per 30 s for 45 min (TCS SP8). Acquired fluorescent, brightfield, and IRM images were merged, and individual cells were manually tracked using MATLAB-based Cell Tracker software (Piccinini et al., 2016 (link)).
5
Quantifying Gap Junctional Coupling
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The extent of gap junctional coupling between MSCs, HSCs, and CMs was measured by FRAP microscopy. For identification, stem cells were labeled with 5 µM Vybrant® CFDA SE cell tracker (Thermo Fisher Scientific) for 15 min before co-cultured with CMs on 4-chamber µ-slides (ibidi). After 4 days cells were loaded with 5 µM calcein red orange (Thermo Fisher Scientific) for 30 min at 37 °C and washed with PBS. 3D-FRAP measurement was performed using ELYRA PS.1 LSM 780 confocal imaging system equipped with a CO2 cage incubator (Carl Zeiss). Images were acquired with 40x/NA 1.3 oil objective. A single MSC within a cluster of CMs was bleached for 50 s by a 561 nm laser beam. Fluorescence recovery was detected every 60 s for 13 min by z-stack acquisition from 15 images. For image analysis maximum projections were created using Zen2011 software (Carl Zeiss). Two unbleached cells were selected as reference to exclude photobleaching artefacts during FRAP measurement. All FRAP measurements were performed on 6–18 cells per experiment and repeated at least 4 times.
6
Fluorescent Labeling in Cell Studies
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7
TGF-β Induced Epithelial-Mesenchymal Transition
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A549 cells (ATCC CCL 185) were grown at 37 °C with 5% CO2 in DMEM (Wako, Lot: AWG7009) with 10% fetal bovine serum (Nichirei Bioscience, Lot 1495557) and penicillin/streptomycin (Wako, Lot 168-23191). At 0 h, 106 cells were seeded in three 10 cm dishes (TRP, Cat. num. 93100). At 24 h, the medium was replaced with DMEM without serum after 3 times washing with PBS (Wako, Lot 045-29795). At 48 h, one-third of the dishes were stimulated by treating with 5 ng/ml TGF-β (R&D systems, USA, Accession #P01137). At 66 h, the second third was stimulated with the same treatment. At 72 h, cells for each treatment duration (0, 6, and 24 h) were stained with combinations of Calcein AM and Calcein red-orange (Thermo Fisher Scientific, L3224 and C34851). After 25 min stain at 37 °C, single cell suspensions were prepared by trypsinization and gentle washing for C1 cell loading. Transcriptome alignment of the C1-positive controls against 79 reference genomes of Mycoplasma or Acholeplasma, including Mycoplasma hominis, confirmed the absence of contamination.
8
C. acnes Interaction with Disc Cells
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NP-derived disc cells (KB7007) were seeded in 6-well plates at a density of 105 cells per well (antibiotic-free) and allowed to attach overnight. A fresh culture of C. acnes type IA1 was fluorescently labelled (in the dark) with 2 mL of 10 μM carboxyfluorescein succinimidyl ester (CFSE) in sterile phosphate buffered saline (PBS) for 1 h at 37 °C with shaking. The bacterial cells were then washed twice in sterile PBS before inoculation of disc cells at MOI = 1:100 and 1:1000 for 24 and 48 h. Images were acquired using a confocal Zeiss LSM 800 microscope with a Plan-Apochromat 40× NA 1.2 W objective (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) and laser lines of 488 nm (CFSE) and 561 nm. The latter detected Calcein Red-Orange (ThermoFisher), which was used (at 1 μM) as a dye for NP cell viability (retained by live cells).
9
Mixed Cell Sequencing with SCOPE-seq2
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Human U87 cells were stained with Calcein AM (ThermoFisher Scientific, cat# C3100MP) and mouse 3T3 cells were stained with Calcein red-orange (ThermoFisher Scientific, cat# C34851) in culture medium at 37 °C for 10 min. The stained cells are then dissociated into single cell suspension by 0.25% Trypsin–EDTA (Life Technologies, cat# 25200-072) and re-suspended in TBS buffer. The U87 and 3T3 cells were mixed at 1:1 ratio with a final total cell concentration 1000 cells/μL.
The mixed cell suspension was processed and sequenced with SCOPE-seq2 workflow described above (PJ070).
Images and sequencing data were processed with the SCOPE-seq2 pipeline described above.
10
Single-cell transcriptome analysis of T cells
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T cells (Jurkat) are cultured in an incubator containing 5% CO2 at 37 °C using Advanced RPMI 1640 (Gibco/Invitrogen) with 10% heat inactivated FBS, 100 units per ml penicillin and 100 μg ml–1 streptomycin. Cells are stained using calcein (25 μM calcein red-orange, AM, C34851, ThermoFisher) in 1× PBS, and incubated on ice for 30 min. Cells are washed twice (HBSS, no calcium, no magnesium, 14170112, ThermoFisher) and re-suspended in 18% OptiPrep Density Gradient Medium (Sigma-Aldrich) in HBSS. Polyacrylamide beads are polymerized with 10 μM Acyridited oligonucleotides (IDT) containing a FAM labeled 3′ end. The flow rate is 4000 μl h–1 50 μm beads, 4000 μl h–1 lysis buffer (0.1% LiDS, 1 mM EDTA, 20 mM TRIS 8.3, 500 mM LiCl), and 8000 μl h–1 oil (Biorad Droplet Generation Oil for EvaGreen #1864005). To quickly find conditions to generate monodispersed bead-triggered drops, a jet is formed in the absence of beads. Drops are collected into a 10 mL syringe for later re-injection and detection. To calculate the speed of drop generation, timetrace data of fluorescence intensity was collected for flowing droplets and analyzed in Matlab using the fast fourier transform (fft) function. The power spectrum was calculated as the square of the absolute value of fft divided by the number of samples.
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