SyEV were stained with DiO (Molecular Probes, Eugene, OR) for 30 min at 37 °C. RAW 264.7 cells labelled with Cellmask Deep Red (Thermo Fisher Scientific, Waltham, MA) were incubated with the fluorescent SyEV for 6 h. The cells were fixed with 4% formaldehyde and then permeabilized with 0.2% Triton X-100 followed by mounting with Prolong Gold antifade reagent (Thermo Fisher Scientific, Waltham, MA). The uptake profile was monitored by a confocal microscope (Zeiss Axio observer; Carl Zeiss, Oberkochen, Germany). For the uptake inhibitor treatment, RAW 264.7 cells were preincubated with Dynasore (Sigma Aldrich, St. Louis, MO) for 30 min and then treated with DiO-SyEV for 6 h. Flow cytometry was performed using a BD FACSVerse Flow Cytometer running BD FACSuit Software (BD Biosciences, San Jose, CA) and FlowJo Software (Tree Star Inc., Ashland, OR).
Facsuit software
Manufactured by BD
Sourced in United States
The BD FACSuit Software is a comprehensive flow cytometry data analysis software developed by BD. It provides a user-friendly interface for the acquisition, visualization, and analysis of flow cytometry data. The software supports a wide range of data file formats and offers various tools for gating, data compensation, and statistical analysis.
Automatically generated - may contain errors
Lab products found in correlation
Facsverse flow cytometer, by BD (12 mentions)
Cellmask deep red, by Thermo Fisher Scientific (6 mentions)
Axio observer, by Zeiss (5 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (4 mentions)
Dynasore, by Merck Group (3 mentions)
Facsverse, by BD (2 mentions)
Annexin 5 fitc, by BD (1 mentions)
Nis element br software, by Nikon (1 mentions)
Facsdiva version 6, by BD (1 mentions)
Facsaria flow cytometer, by BD (1 mentions)
Pi staining buffer, by BD (1 mentions)
Cd3 fitc, by Abcam (1 mentions)
Apc conjugated anti mouse igg, by BD (1 mentions)
Cd122 pe cy7, by BioLegend (1 mentions)
Facscan, by BD (1 mentions)
Annexin 5 fitc pi cell apoptosis detection kit, by Keygen Biotech (1 mentions)
18 protocols using facsuit software
1
Visualizing Extracellular Vesicle Uptake
2
Intracellular ROS Detection by H2DCFDA
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The reduced non‐fluorescent H2DCFDA can be oxidized and converted into fluorescent 2′, 7′‐dichlorofluorescein (DCF) by intracellular ROS. In this protocol, we applied H2DCFDA to label the intracellular ROS and detected the DCF intensity by flow cytometry. Cells were harvested by trypsinization, centrifuged, and washed with PBS and dissolved the pellet in PBS at a density of 1–2 × 106 cells/ml and then DCFDA (Sigma‐Aldrich) was added at the final concentration of 20 μM. The cells were then incubated in dark and prepared for measuring the fluorescence in flow cytometer. The resultant fluorescence intensity was measured with control in BD FACSVerse flow cytometer, USA with blue laser (wave length 488 nm) excitation in FITC channel for resulting DCF fluorescence, and readings were analyzed and represented with BD FACSuit software.
3
Macrophage Interaction with Extracellular Vesicles
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EV or NV were stained with DiO (Molecular Probes) for 30 min at 37 ˚C. Macrophage cell line (RAW 264.7 and MH-S) was labeled with Cellmask Deep Red (Thermo Fisher Scientific) followed by treated with DiO-labeled EV or NV for 6 h. The macrophage cell was fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and observed by a fluorescence light microscope (Zeiss Axio observer; Carl Zeiss). Also, flow cytometry was analyzed using BD FACSVerse Flow Cytometer running BD FACSuit Software (BD Biosciences) and FlowJo Software (Tree Star Inc.).
4
Annexin V-FITC Apoptosis Assay
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After 72 h of X-ray or proton irradiation, the cells were incubated with annexin V-FITC (BD Pharmingen, San Diego, CA, USA) and 2 μg/ml PI in annexin V binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) in the dark for 15 min at 37°C. The apoptotic cell populations were analysed using a BD FACSVerse flow cytometer and a BD FACSuit software.
5
Uptake of Nanovesicles by Macrophages
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NVs were stained with DiO (Molecular Probes, Eugene, OR) for 20 min at 37 °C. Cellmask Deep Red (Thermo Fisher Scientific, Waltham, MA)-labeled RAW 264.7 cells were incubated with DiO-labeled NVs for 6 h. The cells were fixed with 4% paraformaldehyde, and then permeabilized with 0.2% Triton X-100, followed by mounted with Prolong Gold antifade reagent (Thermo Fisher Scientific, Waltham, MA). The uptake was observed by a fluorescence light microscope (Zeiss Axio observer; Carl Zeiss, Oberkochen, Germany). For the uptake inhibitor treatment, RAW 264.7 cells pretreated with dynasore (Sigma Aldrich, St. Louis, MO) for 1 h were sequentially incubated with DiO-NVs for 6 h. Flow cytometry was analyzed using BD FACSVerse Flow Cytometer running BD FACSuit Software (BD Biosciences, San Jose, CA) and FlowJo Software (Tree Star Inc., Ashland, OR).
6
Spleen Cell Immunophenotyping Protocol
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A single cell
suspension of the spleen
from immunized mice was prepared, and 3 × 105 cells/group
were taken in a tube and washed with PBS. Then, 1 μL/tube of
the Fc blocker was added and incubated
for 15 min, followed by washing. Cells were added with fluorochrome-conjugated
anti-CD4, -CD8, and -CD 19 antibodies and incubated for 45 min in
the dark. Cells were washed twice and resuspended in sheath buffer
for flow cytometric analysis. Cells were acquired using FACSVerse
and analyzed using BD FACSuit software.23 (link)
suspension of the spleen
from immunized mice was prepared, and 3 × 105 cells/group
were taken in a tube and washed with PBS. Then, 1 μL/tube of
the Fc blocker was added and incubated
for 15 min, followed by washing. Cells were added with fluorochrome-conjugated
anti-CD4, -CD8, and -CD 19 antibodies and incubated for 45 min in
the dark. Cells were washed twice and resuspended in sheath buffer
for flow cytometric analysis. Cells were acquired using FACSVerse
and analyzed using BD FACSuit software.23 (link)
7
Fluorescent labeling and uptake of OMVs
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OMVs were stained with 5 μM of DiO (Molecular Probes) for 20 min at 37 °C. To observe the cellular uptake of OMVs, DiO-labeled OMVs were treated to Cellmask Deep Red (Thermo Fisher Scientific)-labeled HL-1 grown on coverslips and incubated for 6 h. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and then mounted with Prolong Gold antifade reagent (Thermo Fisher Scientific). A fluorescence light microscope (Zeiss Axio observer; Carl Zeiss) was used for analysis. For the endocytosis inhibitor treatment, HL-1 cells were pretreated with 150 µM dynaosre (Sigma Aldrich) for 1 h at 37 °C, and then incubated with DiO-OMVs for 6 h as described above. Flow cytometric analysis was performed using BD FACSVerse Flow Cytometer running BD FACSuit Software (BD Biosciences) and analyzed with FlowJo Software (Tree Star Inc.).
8
FITC-insulin Liposome Cellular Uptake
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Fluorescence-activated cell sorting analysis (FACS) was used to study the cellular uptake characteristics of TR146 cells. The cells were seeded at a density of 3×105 cells per well in a 12-well plate and incubated for 24 hours in a humidified incubator under 5% CO2 atmosphere at 37°C. Then, the cells were treated with FITC-insulin-loaded liposomes and incubated for 8 hours. Subsequently, the cells were washed twice with HBSS–HEPES buffer (pH 7.4) to remove the traces of liposomal vesicles left in the wells, harvested, and suspended in 0.5 mL of ice-cold FACS buffer (10% FBS and 2% sodium azide in PBS, pH 7.4). The dispersed cells were introduced immediately to FACS analysis using BD FAC suit software (BD Biosciences, San Jose, CA, USA). For the quantification of median fluorescence intensity (MFI) values, 5×103 designated cells were collected per histogram.
9
Vesicle Uptake by Macrophages
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OMV or SyBV were stained with DiO (Molecular Probes, Eugene, OR) for 1 h at 37 °C. MH-S cells labelled with Cellmask Deep Red (Thermo Fisher Scientific, Waltham, MA) were incubated with the DiO-labelled vesicles for 6 h. Flow cytometry was analyzed using a BD FACSVerse Flow Cytometer running BD FACSuit Software (BD Biosciences, San Jose, CA) and FlowJo Software (Tree Star Inc., Ashland, OR).
10
Tracking Virus-BMDC Interactions
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SyBV labelled with DiO (Molecular Probes, Eugene, OR, USA) were incubated with Cellmask Deep Red (Thermo Fisher Scientific)‐labelled BMDCs for 3, 6, or 12 h. The cells were fixed with 4% paraformaldehyde and then permeabilized with 0.2% Triton X‐100. The uptake was analysed by a fluorescence light microscope (Zeiss Axio Observer; Carl Zeiss) following mounting with Prolong Gold antifade reagent (Thermo Fisher Scientific). For the uptake inhibitor treatment, BMDCs pretreated with dynasore (Sigma Aldrich, St. Louis, MO, USA) were incubated with DiO‐labelled SyBV for 6 h. Flow cytometry was analysed using a BD FACSVerse Flow Cytometer running BD FACSuit Software (BD Biosciences, San Jose, CA) and FlowJo Software (Tree Star Inc., Ashland, OR, USA).
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