GYF-21 was isolated from Chinese agarwood and the isolation procedure was described primarily (Huo et al., 2015 (link)). GYF-21 was dissolved at a concentration of 25 mM in dimethyl sulfoxide (DMSO). Cell Counting Kit-8 (CCK-8) was purchased from Beyotime, Co. (Shanghai, China). Lipopolysaccharide (LPS) from Escherichia coli O55: B5 was purchased from Sigma Chemicals, Co. (St. Louis, MO, United States). ELISA kits for determining TNF-α,IL-6, IL-1β, MCP-1, MIP-1α, IFN-γ, and IgG were purchased from R&D Systems (Minneapolis, MN, United States). The antibodies to p65, I-κB, p38, ERK1/2, JNK, STAT1, STAT3, and their phosphorylated forms were purchased from Cell signaling Technology (Beverly, MA, United States). Monoclonal antibodies (mAbs) conjugated to APC, APC, PE, FITC, PE, FITC, FITC, PE, PE-Cy7, Alexa Fluor 647, FITC, and PE (specific for Ly-6G, CD11c, CD11b, CD62L, CD69, CD25, CD80, CD86, CD4, CD8, IFN-γ, and IL-17A), and purified monoclonal antibodies (specific for CD3e, CD28, IFN-γ, and IL-4) were obtained from Becton Dickinson (San Diego, CA, United States). Magnetic bead isolation kit for mouse CD4+ T cells, naive CD4+ T cells and CD8+ T cells were purchased from Miltenyl Biotec (Bergisch Gladbach, Germany). Recombinant mouse GM-CSF, IL-4, IL-12, IFN-γ, IL-6, and TGF-β were purchased from PeproTech (Rocky Hill, NJ, United States).
Fluorescein isothiocyanate (fitc)
Manufactured by BD
Sourced in United States, United Kingdom, Germany, France
FITC (Fluorescein Isothiocyanate) is a fluorescent dye commonly used in various laboratory applications. It is a water-soluble, bright green-yellow fluorescent compound that can be conjugated to a wide range of biomolecules, including proteins, antibodies, and nucleic acids. FITC has excitation and emission wavelengths of approximately 490 and 525 nanometers, respectively.
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Lab products found in correlation
Facscalibur, by BD (20 mentions)
Facscalibur flow cytometer, by BD (17 mentions)
Percp cy5, by BD (15 mentions)
Annexin 5, by BD (11 mentions)
Facscanto 2, by BD (9 mentions)
Propidium iodide, by BD (9 mentions)
Apc cy7, by BD (7 mentions)
Cellquest software, by BD (6 mentions)
Facsuite software, by BD (6 mentions)
Percp cy5, by BioLegend (5 mentions)
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (5 mentions)
Alexa fluor 647, by BD (4 mentions)
Annexin 5 fitc, by BD (4 mentions)
Flow cytometry, by BD (4 mentions)
Facscanto 2 flow cytometer, by BD (4 mentions)
Lsrfortessa, by BD (3 mentions)
Facsaria 2, by BD (3 mentions)
Accuri c6 flow cytometer, by BD (3 mentions)
Phycoerythrin pe, by BD (3 mentions)
Fluorescein isothiocyanate (fitc), by BioLegend (3 mentions)
233 protocols using fluorescein isothiocyanate (fitc)
1
Isolation and Characterization of GYF-21 from Agarwood
2
Multiparametric Flow Cytometry of Immune Cells
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After stimulation, in vitro PBMCs and MCs from AAA lesions were harvested and analyzed by flow cytometry for T cell population phenotypes. Each immunofluorescence reaction was carried out using 3 × 105 cells diluted in PBS. Initially, the cells were incubated with specific antibodies for surface markers: CD3 (FITC, PE (Caltag), and PerCP (BioLegend)), CD8 (APC (BioLegend) and FITC (BD Biosciences)), CCR5 (FITC (BD Biosciences)), CCR6 (PE (BioLegend)), CXCR3 (PE (BD Biosciences)), and CCR4 (PerCP/Cy5.5 (BioLegend)). Next, the cells were fixed in 2% formaldehyde, permeabilized with saponin (0.5% in PBS; Sigma-Aldrich), and subjected to intracellular staining for IFN-γ (FITC—BD Biosciences), TNF-α (PE—BD Biosciences), IL-4 (APC—BioLegend), IL-17 (PE—eBioscience), and IL-22 (eFluor 660—eBioscience, San Diego, CA, USA). The cells were acquired on a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA). The analysis was carried out using FCS Express software (De Novo Software, Glendale, CA, USA).
3
Detecting Apoptosis by Flow Cytometry
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Cells (1 × 106) were seeded in six‐well plates, and after being cultured for 24 hours, they were collected and centrifuged. 5 μL of PI(Propidium Iodide) 5 μL of FITC(Fluorescein Isothiocyanate) (BD Pharmingen, USA, New York) were added after the cells were pelleted and resuspended in 100 μL of 1× binding buffer. Apoptosis was detected by flow cytometry (LSRFortessa, BD, USA, New York) after 15 minutes of incubation in the dark at room temperature. The results were analysed by FlowJo 7.6.2.
4
Bacterial Strain Labeling and Flow Cytometry
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Bacterial strains listed in Supplementary Table S1 were labeled with FITC (Solarbio, Beijing, China). Briefly, FITC was dissolved with DMSO (Sigma-Aldrich, St. Louis, MO) and added to the bacterial suspension (10 9 CFU mL -1 ), then cells were kept at 37 °C for 1.5 h before flow cytometry analysis. The fluorescence signal of FITC-labeled bacteria was detected by the FL1 (FITC/FAM) channel under a 488 nm laser excitation using a flow cytometer (Becton Dickinson, Franklin Lakes, NJ). 30 The fluorescence intensity was analyzed with Photoshop software CS6.
5
Monoclonal Antibodies for Cell Analysis
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Commercially available monoclonal antibodies used in this study were as follows: FITC or PerCP anti-CD4; FITC or APC anti-CD8; APC or PE anti-CD25; FITC anti-CD45.1, anti-CD45.2, anti-Thy1.2, and anti-BrdU; and anti-CD3 (2C11) mAbs (BD Pharmingen, CA). PE or APC anti-Foxp3 antibodies were purchased from eBioscience (CA). Carboxyfluorescein diacetate succinimidyl ester (CSFE) was purchased from Molecular Probes (Invitrogen, CA), fluorescein isothiocyanate (FITC) was purchased from Thermo Fisher Scientific, and 5-bromo-2′-deoxyuridine (BrdU) was purchased from Sigma-Aldrich.
6
HLA-DR Expression in HNSCC Cells
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After pretreatment, HLA‐DR expression in HNSCC cell lines with a MEK1/2 inhibitor (U0126, 3 μM; Promega) for 48 h was evaluated by flow cytometry. Anti‐HLA‐DR Abs conjugated with FITC (BD Pharmingen) and anti‐MHC class I Abs conjugated with FITC (BD Pharmingen) were used. Immunoglobulin G1 (MOPC‐21; BioLegend) and IgG2a (MOPC‐173; BioLegend) were used as isotype controls. Samples were analyzed using a CytoFLEX LX flow cytometer and CytExpert (Beckman Coulter).
7
Immune Cell Profiling in DSS-Induced Mice
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Isolation of immune cells from lamina propria of DSS-treated mice and flow cytometry analysis were conducted as previously described [9 (link)]. Flow cytometry followed routine procedures by using 1 × 106 cells per sample, which were incubated with anti-mouse F4/80, CD4, CD11c, CD80, CD40, and CD86 conjugated with FITC, PE, PERCP, or APC (BD Biosciences, Franklin Lakes, NJ). For the intracellular staining, cells were previously stimulated with PMA and ionomycin for 4 h at 37 °C with the addition of 1 μg/mL Golgi plug. Following extracellular staining, cells were fixed, permeabilised, and stained for IL-10, Foxp3, IL-17, IFN-γ, IL-12, IL-6, IL-4, TGF-β, and IL-23 conjugated with FITC, PE, PERCP, or APC (BD Biosciences, Franklin Lakes, NJ). Flow cytometric analysis was conducted on a BD Biosciences FACSCalibur and analyzed by Flowing Software analysis program.
8
Apoptosis Profiling in Transfected DLBCL
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Transfected DLBCL cells were harvested in binding buffer (Sigma‐Aldrich) followed by PBS wash. annexin V‐FITC and propidium (PI) (Sigma‐Aldrich) were added to incubate with the suspension for 15 min on ice followed by a PBS wash. A flow cytometer (FACSAria II, BD Biosciences, Frankin Lakes, USA) was used to analyze the percentage of different populations of cells: annexin V‐FITC(−) and PI(−) were defined as living cells; annexin V‐FITC(+) and PI(−) cells were defined as early apoptotic cells; annexin V‐FITC(+) and PI(+) were defined as late apoptotic cells.
9
Phenotyping and Sorting of Mesenchymal Stem Cells
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To assess and analyze the surface marker on MSCs, cells were stained with human CD14, CD45, CD49b, CD49d, and HLA-DR (BD Biosciences)-fluorescein isothiocyanate (FITC) antibodies, human CD29, CD44, CD90, CD340, EGFR, HLA-ABC (BD Biosciences), and CD105 (Serotec, Kidlington, UK)-phycoerythrin (PE) antibodies, and human CD49f (BD Biosciences)-Alexa 647 antibody. Isotype controls were matched to the mouse to detect the nonspecific background signal as negative controls. The stained MSCs were determined with a FACSCalibur instrument. To sort using specific markers, MSCs were stained with an EGFR or CD49f monoclonal antibody. Both EGFR and CD49f were sorted to 95% purity using a FACSVantage cell sorting system (BD Biosciences).
10
Curcumin Modulates GRP78 and CHOP in Huh-7 Cells
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Huh-7 cells were treated with curcumin at different times and tested for GRP78 and CHOP abundance. Cells were grown in 6-well plates, trypsinized and then fixed with 4% paraformaldehyde at 4 °C for 40 min and rinsed several times with PBS. Nonspecific binding sites were blocked for 2 h at room temperature with 5% normal SVF (Gibco, ThermoFisher Scientific, Waltham, MA, USA) in 0.1% Triton X-100-PBS. Caco-2 cells were incubated overnight at 4 °C with primary antibodies (1:100 dilutions with blocking buffer) for GRP78 (Cell Signaling, Danvers, MA, USA) or CHOP (Santa Cruz, CA, USA). Cells were then incubated with the appropriate fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate-conjugated secondary antibodies (BD Biosciences, Grenoble, France) for 2 h at 4 °C. Cells were analyzed by flow cytometry using the green (FL1 = 530 ± 30 nM) or red (FL-2 = 585 ± 42 nm) channels. Each experiment was repeated at least four times in duplicate.
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