Draining inguinal lymph nodes from immunized mice were collected and treated as previously described (47 (link), 48 (link)). Briefly, the tissues were treated with collagenase type IV (Worthington) at a concentration of 1 mg/mL for 20 min at a temperature of 37 °C, and then passed through a 100-μm strainer to obtain a single-cell suspension. The resulting single-cell samples were stained with a range of markers including Zombie UV (BUV496, BioLegend 423107), anti-Ly6C (BV780, BioLegend 128041), anti-Ly6G (APC-Cy7, BioLegend 127624), anti-CD19 (BUV395, BD 563557), anti-CD3 (BB700, BD742175), anti-MHCII (AF700, eBioscience 56-5321-82), anti-CD11b (BV650, BioLegend 101239), anti-CD11c (BV421, BioLegend 117330), anti-CD86 (A647, BioLegend 105020), anti-Siglec-F (PE-CF594, BD 562757), anti-CD45 (BV610, BioLegend 103140), anti-CD169 (PE-Cy7, BioLegend 142412), anti-PDCA-1 (BUV563, BD 749275), anti-CD8a (BUV805, BD 612898), anti-CD103 (PE, eBioscience 12-1031-82), anti-NK1.1 (BV510, BioLegend 108738), and anti-F4/80 (BUV737, BD 749283). The cells were analyzed using the BD FACSymphony analyzer located at the Stanford Shared Fluorescence-activated cell sorting (FACS) Facility.
Facsymphony analyzer
Manufactured by BD
The BD FACSymphony analyzer is a flow cytometry instrument designed for advanced cell analysis. It is capable of simultaneous detection of multiple parameters on single cells, allowing for detailed characterization of complex cell populations.
Automatically generated - may contain errors
Lab products found in correlation
Apc cy7, by BioLegend (3 mentions)
Buv496, by BioLegend (3 mentions)
Bv780, by BioLegend (3 mentions)
Bb700, by BD (3 mentions)
Bv610, by BioLegend (3 mentions)
Buv563, by BD (3 mentions)
Buv805, by BD (3 mentions)
Collagenase type 4, by Worthington (3 mentions)
Facsdiva, by BD (2 mentions)
Dcfh da, by Beyotime (1 mentions)
Fcr blocking reagent, by BioLegend (1 mentions)
Red blood cell lysis buffer, by Beyotime (1 mentions)
6 protocols using facsymphony analyzer
1
Profiling Immune Cell Populations in Inguinal Lymph Nodes
2
Comprehensive Immune Cell Profiling of Vaccinated Mice
3
Effects of Cavitation on 4T1 Cells
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To evaluate the effects of cavitation on cells, 4T1 cells were
collected by trypsinization and dispersed in serum-free RPMI-1640
to give a final cell number of 106 cells/mL. Then, 0.1
mL of 1 mg/mL F127-hMSN solution in PBS was added to 0.9 mL of cell
suspension for imaging experiments, and 0.05 mL of the same solution
of F127-hMSN was added to 0.45 mL of cell suspension for HIFU experiments.
Cells were insonated for 2 min for both modalities, as described above.
Then, 4 μL of ethidium homodimer (2 mM) and 2 μL of Calcein
AM (50 μM in dimethyl sulfoxide) solutions were added to 1 mL
of samples and incubated at RT for 15–30 min. Finally, flow
cytometry was performed using a BD FACSymphony Analyzer. The scatter
plot were divided into three regions, as shown inFigure 6 , and the number of events
in each region was recorded to calculate count (%) values.
collected by trypsinization and dispersed in serum-free RPMI-1640
to give a final cell number of 106 cells/mL. Then, 0.1
mL of 1 mg/mL F127-hMSN solution in PBS was added to 0.9 mL of cell
suspension for imaging experiments, and 0.05 mL of the same solution
of F127-hMSN was added to 0.45 mL of cell suspension for HIFU experiments.
Cells were insonated for 2 min for both modalities, as described above.
Then, 4 μL of ethidium homodimer (2 mM) and 2 μL of Calcein
AM (50 μM in dimethyl sulfoxide) solutions were added to 1 mL
of samples and incubated at RT for 15–30 min. Finally, flow
cytometry was performed using a BD FACSymphony Analyzer. The scatter
plot were divided into three regions, as shown in
in each region was recorded to calculate count (%) values.
4
Profiling Neutrophil ROS Levels
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Neutrophils were analyzed ex vivo for their reactive oxygen species (ROS) level. MHCIIhi or MHCIIlow neutrophils derived from the lungs of tumor-bearing mice were sorted as described in the flow cytometry methods section. Then, the cells were washed and resuspended in serum-free RPMI-1640 medium, and 10 μM DCFH-DA (Beyotime Biotechnology, catalog no. S0033S) was added for 20 min at 37 °C. Cells were washed with PBS three times to remove DCFH-DA that did not enter the cells. Then, the cells were resuspended in PBS, and the activated DCF signal was analyzed in the FITC channel on a BD FACSymphony analyzer (BD Biosciences). Rosup was added to cells and served as a positive control.
5
Comprehensive Immune Cell Profiling of Vaccinated Mice
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Draining iliac lymph nodes from BNT162b2 immunized mice, draining inguinal lymph nodes from YF-17D immunized mice, along with whole lung or spleen, were harvested and digested with 1 mg/mL collagenase type IV (Worthington) for 20 min at 37 °C, followed by smashing with a 100 μm strainer to make a single-cell suspension. Red blood cells from the lung and spleen were lysed before staining. Single-cell samples were then stained with Zombie UV™ (1:300, BUV496; BioLegend #423107), anti-Ly6C (1:500, BV780; BioLegend #128041), anti-Ly6G (1:400, APC-Cy7; BioLegend #127624), anti-CD19 (1:100, BB700; BD #566411), anti-CD3 (1:100, BB700; BD #742175), anti-MHCII (1:400, AF700; eBioscience #56-5321-82), anti-CD11b (1:300, BV650; BioLegend #101239), anti-CD11c (1:400, BV421; BioLegend #117330), anti-CD86 (1:300, A647; BioLegend #105020), anti-Siglec-F (1:400, PE-CF594; BD #562757), anti-CD45 (1:200, BV610; BioLegend #103140), anti-CD169 (1:200, PE-Cy7; BioLegend #142412), anti-PDCA-1 (1:200, BUV563; BD #749275), anti-CD8a (1:200, BUV805; BD #612898), anti-CD103 (1:100, PE; eBioscience #12-1031-82), anti-NK1.1 (1:200, BV510; BioLegend #108738), and anti-F4/80 (1:100, BUV737; BD #749283). Data was collected on a BD FACSymphony analyzer with BD FACSDiva (v8.0.1).
6
Isolation and Analysis of Murine Lung Cells
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The lung tissues were homogenized in RPMI-1640 and 10% FBS containing 0.2 mg/ml collagenase type I/IV and single-cell suspensions were generated from lung tissues using a gentle MACS™ Octo Dissociator with Heaters following the manufacturer’s protocol (Miltenyi Biotec). The digested lung tissues were filtered through 70 μM cell strainers and red blood cells were lysed using red blood cell lysis buffer (Beyotime). The resulting single-cell suspensions were washed with PBS and resuspended in PBS containing 1% FBS. The cell suspensions were incubated with FcR Blocking Reagent (Biolegend) for 15 min at 4 °C, stained with fluorescent conjugated Abs for 30 min at 4 °C and washed twice with PBS. Positive staining with the BD fixable viability dye FVS620 (BD Biosciences) was used to exclude dead cells. Data were collected on a BD FACSymphony analyzer (BD Biosciences) and analyzed with FlowJo software (v.10.4).
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