PerCP/Cy5.5 α-mouse CD3 antibody (17A2), APC/Cy7 α-mouse CD4 antibody (RM4-5), PE/Cy7 α-mouse CD8a antibody (53–6.7), FITC α-mouse/human Helios antibody (22F6), biotin α-mouse/human CD44 antibody (IM7), PE/Cy7 α-mouse CD25 antibody (PC61), biotin α-mouse H-2Dd antibody (34-2-12), FITC α-mouse/human CD11b antibody (M1/70), PE α-mouse CD19 antibody (6D5), FITC α-mouse NK-1.1 antibody (PK136) and PE α-mouse CD62L antibody were purchased from BioLegend (San Diego, CA). α-mouse Neuropilin-1 PE (761705) was acquired from R&D Systems (Minneapolis, MN). α-mouse/rat Foxp3 APC (FJK-16s) and α-mouse/rat Ki-67 PE-Cy7 (SolA15) were obtained from eBioscience. For intracellular staining the cells were permeabilized with the Foxp3/Transcription Factor Staining Buffer Set from eBioscience according to the manufacturer’s specification. Flow cytometric analysis was performed with a BD FACSCanto II or Beckman Coulter FC500 flow cytometer and data were analyzed by FlowJo (10.0.8) software.
Fc500
Manufactured by FlowJo
Sourced in United States
The FC500 is a flow cytometer designed for cell analysis and sorting. It is a compact, benchtop instrument that utilizes laser technology to detect and analyze the physical and fluorescent characteristics of individual cells passing through a focused laser beam. The FC500 is capable of measuring multiple parameters, including size, granularity, and the expression of fluorescently labeled cellular markers.
Automatically generated - may contain errors
Lab products found in correlation
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (1 mentions)
Rnase a, by Merck Group (1 mentions)
70 μm cell strainer, by Jet Biofil (1 mentions)
Cell cycle staining kit, by Beyotime (1 mentions)
Annexin 5 fitc pi apoptosis kit, by Beyotime (1 mentions)
Fc500, by Beckman Coulter (1 mentions)
Fixation and permeabilization buffer, by BD (1 mentions)
Leukocyte activation cocktail, by BD (1 mentions)
Fitc anti human cd80, by BioLegend (1 mentions)
Rnase a, by Solarbio (1 mentions)
7 protocols using fc500
1
Multiparametric Immune Cell Profiling
2
Cell Cycle Analysis by Flow Cytometry
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One million cells were harvested after 48 hr of transfection, washed with PBS and then fixed in 50% ethanol. Cells were treated with 2.5 μL RNase A (20 mg/mL; Sigma-Aldrich) for 1 hr at 37°C. Ten microliters of PI (1 mg/mL; Sigma-Aldrich) was added into cell suspension and mixed gently. PI staining was determined by flow cytometry (Cytomics FC 500) and further analyzed by FlowJo software version 7.6.2. All experiments were performed independently in triplicate.
3
Quantifying Immune Cell Subsets
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Single-cell suspensions were generated from the spleen and blood collected from experimental mice. After red blood cells were eliminated by using the Red Cell Lysis buffer solution (BD Biosciences, San Jose, CA, USA), lymphocytes were filtered through a 70-μm cell strainer (Jet Biofil, Guangzhou, China) and diluted in 1× PBS (Thermo Fisher Scientific, Waltham, MA, USA). T cells (CD3e+), B cells (CD3e−CD45R/B220+), NK cells (CD3e−CD49b+), effector T cells (CD45+CD3e+CD44+), and effector B cells (CD45+CD3e−, CD45R/B220+CD138+CD27+) in the spleen and blood were directly quantified using flow cytometry with monoclonal antibodies (1 μg/ml) directed against the noted specific human and mouse antigens (Table 2 ; all antibodies purchased from BD Pharmingen, San Diego, CA, USA). The stained cells were detected using a flow cytometer (Beckman FC-500, Miami, FL, USA) and analyzed with FlowJo 7.6.1. software.
4
Cell Cycle and Apoptosis Analysis
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Cell cycle analysis was performed using a cell cycle staining kit (Beyotime, Cat# C1052) following the manufacturer’s instructions. Cells were fixed with 70% cold ethanol for 3 h, rinsed using PBS, and then labeled with propidium iodide in the presence of RNase A for 30 min in the dark. Flow cytometry (Beckman Coulter FC500, USA) and FlowJo software (version 7.6.1) were used to determine the percentages of cells in G0/G1, S, and G2/M phases. Apoptosis was analyzed using an Annexin V-FITC/PI apoptosis kit (Beyotime, Cat# C1062M) according to the manufacturer’s instruction. Treated cells were harvested and rinsed twice with cold PBS before staining with 5 μL of Annexin V‐FITC and 5 μL of propidium iodide (PI). They were then incubated in the dark for 15 min at room temperature before analysis by flow cytometry (Beckman Coulter FC500, USA). The PHAGF apoptotic rate was determined by the proportion of Annexin V-FITC positive cells (early apoptosis) plus Annexin V-FITC/PI positive cells (late apoptosis). Cells negative for Annexin V-FITC and PI were considered live cells. Assays were done in triplicate.
5
Th17 Cell Quantification by Flow Cytometry
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All flow cytometry analyses were performed after 72 h post-polarization. For Th17 detection, cells were first re-stimulated with Leukocyte Activation Cocktail (BD Pharmingen, 550583) for intracellular cytokine staining. After 4 h, cells were then washed, fixed/permeabilized with fixation and permeabilization buffers (BD Pharmingen, 554722) and stained with mouse CD4-FITC (145-2C11), mouse IL-17A-PE (TC11-18H10) (all for BD Pharmingen). For analysis and gating, we set up auxiliary staining groups including no staining, single staining, isotype staining. Finally, data were acquired on flow cytometry (Beckman, FC-500) and analyzed by the FlowJo V10 software.
6
Macrophage Surface Marker Analysis
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To analyze surface marker expression,
macrophages were treated for 24 h with IONPs in a medium supplemented
with different biological sera and they were then stained with: BIOT-anti-mouse
CD86 (BIOLEGEND, b128959); PE-anti-mouse CD80 (PARMIGEN, 5533759);
PE-anti-human CD86 (IMMUNOTECH, IM2729); and FITC-anti-human CD80
(BIOLEGEND, 305206). The data were acquired on an FC500 flow cytometer
and analyzed with the FlowJo software.
macrophages were treated for 24 h with IONPs in a medium supplemented
with different biological sera and they were then stained with: BIOT-anti-mouse
CD86 (BIOLEGEND, b128959); PE-anti-mouse CD80 (PARMIGEN, 5533759);
PE-anti-human CD86 (IMMUNOTECH, IM2729); and FITC-anti-human CD80
(BIOLEGEND, 305206). The data were acquired on an FC500 flow cytometer
and analyzed with the FlowJo software.
7
Cell cycle analysis of HUVECs
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HUVECs were treated with different concentrations of GE or Bpv to analyze the cell cycle when growing to a density of 50%. The cells were digested and xed in 70% ethanol overnight at 4 o C. The cells were resuspended in PBS the next day, and stained with 400 µL propidium iodide (PI) solution (50 µg/mL) (Solarbio, P8080, Beijing, China) and 100 µL RNase A (100 µg/mL) (Solarbio, R1030, Beijing, China) for 30 min in the dark. Finally, it was analyzed by ow cytometry (Beckman Coulter, FC500) and FlowJo software (Version 10.6.0).
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