CD38+ NK cells and autologous Treg cells were isolated from the peripheral blood of RA patients using flow cytometry-based methods, as described above. CD38+ NK cells were incubated in 50 μM C3G or an equal volume of PBS at 37 °C for 24 h. Isolated Treg cells were labeled with 5 μM carboxy fluorescein diacetate succinimidyl ester (CFSE; BioLegend) for 10 min at 37 °C. The pretreated CD38+ NK cells and Treg cells were washed and seeded at an effector-to-target cell (E:T) ratio of 20:1 in a 96-well U-bottom plate for 4 h at 37 °C. 7-Aminoactinomycin D (7-AAD; 5 μg/ml; BioLegend) was added to the culture, and the mixture was incubated for 20 min at 4 °C. Cell death was measured using flow cytometry. The percentage of specific lysis was calculated based on the following formula: 100 × (CFSE+ 7-AAD+ Treg cells/total number of CFSE+ Treg cells). The flow cytometry-based cytotoxicity assay for CD38+ NK cells against Treg cells was designed following a previously reported method [37 (link), 38 (link)].
Carboxyfluorescein diacetate succinimidyl ester cfse
Manufactured by BioLegend
Sourced in China
Carboxyfluorescein diacetate succinimidyl ester (CFSE) is a fluorescent dye used for cell tracking and proliferation assays. It covalently binds to primary amines within cells, allowing for the monitoring of cell division and migration.
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Lab products found in correlation
7 aminoactinomycin d, by BioLegend (1 mentions)
F ab 2 anti igm antibody, by Jackson ImmunoResearch (1 mentions)
Cpg oligodeoxynucleotide, by InvivoGen (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by BioLegend (1 mentions)
Cd19 pe cy7 clone hib19, by BioLegend (1 mentions)
Zombie aqua amcyan viability dye, by BioLegend (1 mentions)
Cd20 percp cy5.5 clone 2h7, by BioLegend (1 mentions)
Dextran sulfate sodium (dss), by MP Biomedicals (1 mentions)
Collagenase type 4, by Merck Group (1 mentions)
Hyaluronidase, by Merck Group (1 mentions)
Deoxyribonuclease, by Merck Group (1 mentions)
Isotype control monoclonal antibodies, by BioLegend (1 mentions)
Mf 14, by BioLegend (1 mentions)
4 protocols using carboxyfluorescein diacetate succinimidyl ester cfse
1
Evaluating CD38+ NK Cell-Mediated Treg Cytotoxicity
2
B cell proliferation upon DENV-2 infection
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B cells isolated from healthy donors were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (Biolegend) and either infected with DENV-2 at an MOI of 5 or remained uninfected. After 2 h of inoculation, cells were washed to remove the inoculum and stimulated with CpG oligodeoxynucleotides (1 μg/ml; Invivogen, San Diego, CA, USA) and F(ab′)2 anti-IgM antibody (4 μg/ml; Jackson ImmunoResearch, PA, USA) or remained unstimulated. Cells were cultured in RPMI supplemented with 10% FBS for 6 days. The cells were then harvested and stained with Zombie Aqua (AmCyan) viability dye (BioLegend) followed by CD19 PE/Cy7 (clone HIB19) and CD20 PerCp/Cy5.5 (clone 2H7) to identify live B cells and analyzed for the expression of CFSE in the FITC channel. Proliferation was measured as the percentage of B cells with decreased intensity of CFSE compared to unstimulated B cells.
3
Phenotyping Immune Cells by Flow Cytometry
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Recombinant mouse Fms-like tyrosine kinase 3 (Flt3) ligand was purchased from Peprotech (Suzhou, China). Fluorescein-conjugated monoclonal antibodies against CD45 (30 -F11), CD11c (N418 ), CD11b (M1/70 ), adhesion G protein-coupled receptor E1 (F4/80, BM8 ), Gr-1, programmed cell death ligand 1 (PD-L1, 10 F .9G2 ), CD3 (17A2 ), CD4 (GK1.5 ), CD8 (53 – 6.7 ), IFNγ (XMG1.2 ), and FOXP3 (MF-14">MF-14 ); isotype control monoclonal antibodies, and carboxyfluorescein diacetate succinimidyl ester (CFSE) were purchased from Biolegend (Beijing, China); leukocyte activation cocktail (LAC) was purchased from BD Pharmingen (Shanghai, China). Microbead-conjugated monoclonal antibodies against CD8 or CD11c were purchased from Miltenyi Biotec (Shanghai, China). The iTAg Tetramer/PE-H-2Kb OVA (SIINFEKL) detection kit was purchased from MBL (Shanghai, China). Ovalbumin peptide SIINFEKL (OVA257-264), collagenase type IV, hyaluronidase, and deoxyribonuclease were purchased from Sigma-Aldrich (Shanghai, China). Dextran sulfate sodium (DSS; molecular mass 36,000–50,000 Da) was purchased from MP Biomedicals (Beijing, China). Monoclonal antibodies against human CD8 (EP1150Y) and HDAC9 (EPR5223) were purchased from Abcam (Shanghai, China).
4
Isolation and CFSE Labeling of Mouse Splenic Lymphocytes
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Mouse spleens were harvested under aseptic conditions, washed with PBS and then cut into pieces in DMEM. After the cell suspensions were filtered through a 40 µm membrane, mouse spleen single-cell suspensions were prepared. Spleen lymphocytes were obtained according to the instructions of the mouse spleen lymphocyte isolation kit (Haoyang Biological Manufacture) and centrifuged at 2000 rpm for 10 min. The cell pellets were washed twice with PBS. Mouse splenic lymphocytes were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) (BioLegend). 1.0–10×107 cells splenic lymphocytes were resuspended in 5 µM concentration CFSE working solution. The suspension was protected from light and incubated at 37°C for 20 min. DMEM with 10% FBS was added to terminate staining. After centrifugation at 2000 rpm for 10 min, the cells were resuspended in preheated cell culture medium and incubated in the cell incubator for subsequent experiments. Splenic lymphocytes labeled with CFSE were used in subsequent experiments.
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