Virus lipid labeling using 3,3′-dioctadecyl-5,5′-di(4-sulfophenyl)oxacarbocyanine (SP-DiOC18; Molecular Probes catalog no. D7778) and octadecyl rhodamine b chloride (R18; Molecular Probes catalog no. O246) was described previously (25 (link), 26 (link)) and was adapted to the single use of SP-DiOC18 or 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine,4-chlorobenzenesulfonate (DiD; Molecular Probes catalog no. D7757). In brief, IAV (PR8M) was labeled with SP-DiOC18 and R18 at final concentrations of 0.2 µM and 0.4 µM, respectively, or with SP-DiOC18 or DiD alone at 0.2 µM for 1 h at room temperature and was subsequently filtered through a 0.22-µm-pore-size filter (Millipore). Cells were infected with the labeled virus for 30 min on ice, washed twice with PBS, and subsequently incubated for the indicated periods of time at 37°C. Cells were harvested by Accutase (Millipore catalog no. SCR005) treatment and were fixed with 2% formaldehyde for 20 min. Lipid mixing was quantified by FACS analysis, with 10,000 cells analyzed per sample. Linear regression analysis (GraphPad Prism) was used to determine the best-fit linear regression and to calculate the slopes.
Sp dioc18
Manufactured by Thermo Fisher Scientific
Sourced in United States
The SP-DiOC18 is a fluorescent probe that labels the plasma membrane of cells. It is a lipophilic cation that preferentially accumulates in the mitochondria of cells due to the negative membrane potential. The probe can be used to visualize the plasma membrane and mitochondria of cells.
Automatically generated - may contain errors
Lab products found in correlation
510 meta confocal microscope, by Zeiss (2 mentions)
Slowfade, by Thermo Fisher Scientific (2 mentions)
Celltracker green cmfda, by Thermo Fisher Scientific (2 mentions)
Sp diic18, by Thermo Fisher Scientific (2 mentions)
Stereo discovery v20 macro stereo, by Zeiss (2 mentions)
Dulbecco modified eagle medium (dmem), by Corning (2 mentions)
Facscanto 2, by BD (2 mentions)
Sp dioc18, by Merck Group (1 mentions)
0.22 m pore size filter, by Merck Group (1 mentions)
Accutase, by Merck Group (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Matrigel basement membrane matrix, by BD (1 mentions)
Cm dii, by Thermo Fisher Scientific (1 mentions)
Fv3000, by Leica (1 mentions)
Vt1000s vibratome, by Leica (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Evos cell imaging system, by Thermo Fisher Scientific (1 mentions)
0.22 μm pore filter, by Merck Group (1 mentions)
12 protocols using sp dioc18
1
Virus Lipid Labeling Methodology
2
Measuring Viral Fusion Using Fluorescent Dyes
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Measurement of viral fusion was performed according to a protocol previously described (43 (link), 44 (link)). Briefly, IAV A/PR/8/34 was labeled using two fluorescent dyes, R18 (octadecyl rhodamine B chloride) and SP-DiOC18 [3,3′-dioctadecyl-5,5′-di(4-sulfophenyl)oxacarbocyanine] (Life Technologies), at a ratio of 1:2 with final concentrations of R18 of 22 µM and SP-DiOC18 of 46 µM. After intense vortexing for 60 min at RT, labeled virus was filtered through a 0.22-µm-pore-size filter. Virus was bound to cells for 30 min at a cold temperature (4°C), and after the cells were washed with PBS, the temperature was shifted to 37°C for 0, 90, or 180 min. Unfixed and unpermeabilized samples were mounted. Image analysis was carried out using the spot analysis function of ImageJ for SP-DiOC18 staining with a distinct spot size of 20 pixels and a subsequent correction for cell numbers.
3
Viral Internalization Assay in A549 Cells
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A549 cells were transfected with siRNAs and infected with rPR8-GFP virus at an MOI of 100 at 4 °C for 1 hour. The virus had been previously labeled with fluorescent dyes R18 and SP-DiOC18 (Life technologies). After washing cells with cold PBS, serum-free media was added and cells were incubated for 1.5 hours at 37 °C. Cells were then fixed with 4% PFA containing 2.5 μg/ml of Hoechst 33342 before microscopic observation.
4
Optimized Recellularization of Porcine ECM
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The optimized reseeding method was applied using fluorescently labeled MDCKs to track cells in the ECM structure using three-dimensional (3D) imaging. On the day of recellularization, the detached cells were suspended in 1× DMEM (Gibco by Life Technology) containing 5 µM SP-DiOC18 (Life Technologies, Grand Island, NY, USA) and incubated at 37°C for 20 min. Subsequently, the cells were washed three times with 1× DMEM and suspended in cell culture medium for recellularization. The recellularization of whole porcine ECM with labeled cells was performed through ureteral injection while 40 mmHg vacuum was applied to the kidney chamber. After 7 days of cell growth, random slices of renal cortex and medulla were excised and fixed in 4% PFA overnight. Images of the 3D structure of recellularized ECM were obtained using an Olympus FluoView™ FV1000 (Center Valley, PA, USA) laser scanning confocal microscope.
5
Fluorescent Labeling of Cultured Cells
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Example 4
In a subset of studies, RAECs were labeled with DiI or DiO on the day of recellularization. Briefly, media was removed from a confluent plate of RAECs and replaced with DPBS containing 5 μM SP-DiIC18 or SP-DiOC18 (Invitrogen). After 5 minutes of incubation at 37° C., plates were transferred to a refrigerator and incubated for 15 minutes at 4° C. Plates were then washed once with PBS and allowed to recover for 2 hours at 37° C. in culture media before isolation and construct seeding. At the end of the experiment, constructs were removed from the bioreactor and imaged on a Stereo Discovery V20 Macro Stereo (Carl Zeiss Inc.), dissected, placed in Slowfade (Invitrogen) and imaged on a 510 Meta Confocal microscope (Carl Zeiss Inc.).
In separate studies, RAECs seeded constructs were labeled with Cell Tracker Green CMFDA (Invitrogen) on the last day of culture (Day 7), by removing the complete culture media and circulating serum free CMFDA containing DMEM (Cellgro) for 45 minutes at 37° C. CMFDA containing media was then replaced with complete MCDB-131 and the constructs were incubated for 45 minutes. CMFDA-labeled constructs were then removed from the bioreactor, dissected, placed in Slowfade (Invitrogen) and imaged on a 510 Meta Confocal microscope (Carl Zeiss Inc.).
6
SARS-CoV-2 Particle Labeling and Analysis
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SARS-CoV-2 particles were labelled with the self-quenching dye SP-DiOC18 (Invitrogen). SP-DiOC18 was added to 1 ml of virus stock containing approximately 2.5 × 107 PFU/ml at a final concentration of 0.2 μM. The tube was protected from light, incubated while rolling for 1 h at room temperature and subsequently stored at −80°C or used as required. Cells were incubated for 1.5 h with the labelled virus, harvested with trypsin, fixed with 4% PFA for 30 min and analysed by FACSCanto II (BD Biosciences) using the 488 nm laser for excitation.
7
In vitro Angiogenesis Assay Protocol
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The in vitro angiogenesis assay was performed according to a previously published protocol
[31 (link)].
Briefly, 24-well cell culture plates were coated with Matrigel Basement Membrane Matrix (BD Biosciences, Le Pont de Claix France). The culture plates were incubated at 37°C for at least 30 minutes to allow the basement membrane to form a gel. HUAEC were labeled with SP-Dioc18 (3,3’-dioctadecyl-5-5’-di(4-sulfophenyl) oxacarbocyanine, Invitrogen) green dye (2 μg/ml) and VSMC were labeled with CM-Dil red dye (1 μg/ml) (Invitrogen). HUAEC were added on top of the Matrigel matrix (3.104 per well) and then incubated overnight in EBM 10% FCS to induce tube formation. VSMC were then added to the endothelial network (1.5×104 VSMC per well) and the samples incubated for 5 hours. Cells were visualized by inverted-phase fluorescence microscopy (Zeiss, Le Pecq, France). Photographs of ten representative fields were taken and quantified using Histolab software (Microvision, Evry). Statistical analyses were performed using either Student’s t test or ANOVA.
[31 (link)].
Briefly, 24-well cell culture plates were coated with Matrigel Basement Membrane Matrix (BD Biosciences, Le Pont de Claix France). The culture plates were incubated at 37°C for at least 30 minutes to allow the basement membrane to form a gel. HUAEC were labeled with SP-Dioc18 (3,3’-dioctadecyl-5-5’-di(4-sulfophenyl) oxacarbocyanine, Invitrogen) green dye (2 μg/ml) and VSMC were labeled with CM-Dil red dye (1 μg/ml) (Invitrogen). HUAEC were added on top of the Matrigel matrix (3.104 per well) and then incubated overnight in EBM 10% FCS to induce tube formation. VSMC were then added to the endothelial network (1.5×104 VSMC per well) and the samples incubated for 5 hours. Cells were visualized by inverted-phase fluorescence microscopy (Zeiss, Le Pecq, France). Photographs of ten representative fields were taken and quantified using Histolab software (Microvision, Evry). Statistical analyses were performed using either Student’s t test or ANOVA.
8
Mopeia Virus Labeling and Infection
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Mopeia virus particles were labelled with the self-quenching dye SP-DiOC18 (Invitrogen). SP-DiOC18 was added to 1 ml of MOPV virus stock at 1 × 106 PFU/ml at a final concentration of 0.2 μM. The mixture was protected from light and incubated with gentle rolling for 1 h at room temperature. For the heat inactivated control virus was incubated at 75°C for 30 min and cooled to room temperature before labelling. The labelled virus preparations were passed through a 0.45 μM filter before use and diluted in serum-free DMEM for infection of A549 monolayers at an MOI of 5. Cells were incubated with labelled virus at 4°C for 15 min and then incubated for 1.5 h at 37°C after infection. Cells were then harvested with trypsin, fixed with 4% PFA for 10 min and then analysed by flow cytometry on the FACSCanto II (BD Biosciences) using the 488 nm laser for excitation.
9
Fluorescent Labeling of Cultured Cells
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Example 4
In a subset of studies, RAECs were labeled with DiI or DiO on the day of recellularization. Briefly, media was removed from a confluent plate of RAECs and replaced with DPBS containing 5 μM SP-DiIC18 or SP-DiOC18 (Invitrogen). After 5 minutes of incubation at 37° C., plates were transferred to a refrigerator and incubated for 15 minutes at 4° C. Plates were then washed once with PBS and allowed to recover for 2 hours at 37° C. in culture media before isolation and construct seeding. At the end of the experiment, constructs were removed from the bioreactor and imaged on a Stereo Discovery V20 Macro Stereo (Carl Zeiss Inc.), dissected, placed in Slowfade (Invitrogen) and imaged on a 510 Meta Confocal microscope (Carl Zeiss Inc.).
In separate studies, RAECs seeded constructs were labeled with Cell Tracker Green CMFDA (Invitrogen) on the last day of culture (Day 7), by removing the complete culture media and circulating serum free CMFDA containing DMEM (Cellgro) for 45 minutes at 37° C. CMFDA containing media was then replaced with complete MCDB-131 and the constructs were incubated for 45 minutes. CMFDA-labeled constructs were then removed from the bioreactor, dissected, placed in Slowfade (Invitrogen) and imaged on a 510 Meta Confocal microscope (Carl Zeiss Inc.).
10
Two-color Somite Fate Mapping in Skate Embryos
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Two-color somite fate mapping experiments were performed as described in Criswell et al. (2017b) (link) and Ward et al. (2017) (link). S24 skate embryos were removed from their egg cases to a petri dish and anesthetized in tricaine (MS-222 1 mg/L in seawater). Adjacent somites were injected with the red-fluorescent lipophilic dye CM-DiI and the green-fluorescent lipophilic dye SpDiOC18 (ThermoFisher). Concentrated stocks of CM-DiI (5 µg/µL in absolute ethanol) and SpDiOC18 (2.23 µg/µL in dimethylformamide) were diluted 1:10 in 0.3 molar sucrose for injection. After injection embryos were returned to their egg cases and maintained in a flow-through seawater table at 15°C for 8–12 weeks post-injection.
Injected skate embryos were euthanized using an overdose of tricaine (1 g/L in seawater) and fixed in 4% paraformaldehyde overnight at 4°C. Embryos were then rinsed 3 × 5 min in phosphate-buffered saline (PBS), embedded in 15% gelatin in PBS and post-fixed in 4% paraformaldehyde in PBS for 4 nights at 4°C before vibratome sectioning. A Leica VT1000S vibratome was used to cut 100 µm sections of tissue in sagittal plane, which were then DAPI-stained (1 µg/mL), coverslipped with Fluoromount-G (Southern Biotech) and imaged on an Olympus FV3000 (trunk and transitional vertebrae) or Leica Sp5 (tail vertebrae) confocal microscope.
Injected skate embryos were euthanized using an overdose of tricaine (1 g/L in seawater) and fixed in 4% paraformaldehyde overnight at 4°C. Embryos were then rinsed 3 × 5 min in phosphate-buffered saline (PBS), embedded in 15% gelatin in PBS and post-fixed in 4% paraformaldehyde in PBS for 4 nights at 4°C before vibratome sectioning. A Leica VT1000S vibratome was used to cut 100 µm sections of tissue in sagittal plane, which were then DAPI-stained (1 µg/mL), coverslipped with Fluoromount-G (Southern Biotech) and imaged on an Olympus FV3000 (trunk and transitional vertebrae) or Leica Sp5 (tail vertebrae) confocal microscope.
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