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Anticancer Properties of Silver Nanoparticles from Root Extract of Trigonella Foenum-Graecum
Published in Megh R. Goyal, Preeti Birwal, Santosh K. Mishra, Phytochemicals and Medicinal Plants in Food Design, 2022
Ramasamy Harikrishnan, Lourthu Samy S. Mary, Gunapathy Devi, Chellam Balasundaram
The ΔΨ assay was performed by BDTM MitoScreen kit manufacturing by BD Biosciences. Cationic dye, JC-1 is highly sensible to ΔΨ which gathers in mt-polarized membranes part. A 100 µL working JC-1 solution and 100 µL of cell suspension added in each flow cytometry tube, incubated for 15 min at 37 °C containing with 5% CO2. Then 100 µL JC-1 wash buffer was added in each tube. Around 50,000 events were gated to exclude cellular debris by FACS Calibur flow cytometer (CellQuest PRO v4.02 software/ FlowJo v7.1 software) for mt depolarization and the results were stated as % of total events.
Intra-cytoplasmic Cytokine Staining (ICS): Optimizing antigen stimulation for measuring M. tuberculosis-specific T cell response
Published in Ade Gafar Abdullah, Isma Widiaty, Cep Ubad Abdullah, Medical Technology and Environmental Health, 2020
PBMC were stained with fluorochrome-conjugated monoclonal antibodies against surface markers and cytokines following antigen stimulation. Cells were incubated with BV605-conjugated anti-CD3, APC-conjugated anti-CD4 (Biolegend), and live/dead fixable blue staining (Life Technologies) in FACS wash for 30 minutes on ice. The cells were washed, fixed and permeabilized with Cytofix (BD Biosciences) for 20 minutes on ice. The cells were washed with PERM wash buffer (BD Biosciences) prior to staining with FITC-conjugated anti-IFN-γ, AF700-conjugated anti-TNF AF700, and PE-conjugated anti-IL-2 in PERM wash buffer for 30 minutes on ice. After washing with PERM wash, cells were resuspended with FACS wash and 10% neutral buffered formalin. Stained cells were acquired using LSR Fortessa flow cytometer (BD) and the data was analyzed with FlowJo (Tree Star) and FlowJo Boolean gating tool.
Lysosomal Vitamin B12 Trafficking
Published in Bruno Gasnier, Michael X. Zhu, Ion and Molecule Transport in Lysosomes, 2020
Sean Froese, Matthias R. Baumgartner
To prepare the samples for flow cytometry, 36‒72 hours after transfection, cells are harvested by trypsinization and pelleted at 250 × g for 5 min. The cell pellet is washed with 5 mL HBSS, resuspended in 500‒750 µL HBSS, transferred to flow-cytometry compatible tubes through a cell strainer snap cap (e.g. Corning #352235) and kept in the dark until measurement. This cell suspension, containing 0.5‒3 × 106 cells, is added to the flow cell of the flow-cytometer and measured according to the manufacturer’s instructions. Based on a FACSAriaIII (BD Bioscience), we suggest to excite GFP with the 488-nm laser and measure GFP fluorescence with a 540/30 filter and any occurring FRET-signal with a 695/40 filter. To confirm the presence of the fRFP-tagged protein, fRFP can be separately excited with a 561-nm laser and measured with a 610/20 filter (where available). Approximately 30,000 events for each sample should be collected and analyzed using appropriate software (e.g. FlowJo, BD Bioscience).
CDK4/6 blockade provides an alternative approach for treatment of mismatch-repair deficient tumors
Published in OncoImmunology, 2022
Inken Salewski, Julia Henne, Leonie Engster, Paula Krone, Bjoern Schneider, Caterina Redwanz, Heiko Lemcke, Larissa Henze, Christian Junghanss, Claudia Maletzki
Blood was taken routinely from anesthetized mice (retrobulbar venous plexus). Spleen and tumor tissues were dissociated. Single cells were stained with a panel of conjugated monoclonal antibodies (mAb, 0.125 μg to 1.5 μg each). Zombie NIR™ Fixable Viability Kit by Biolegend (San Diego, United States) staining was performed following the protocol Zombie NIR™ Fixable Viability Kit by Biolegend, extracellular staining was performed following the protocol BD Horizon Briliant Stain Buffer (BD Bioscience), followed by lysis and intracellular staining using the protocol of True-Nuclear™ Transcription Factor Buffer Set by Biolegend. Measurements were performed on a spectral flow cytometer (Cytek™ Aurora). For extracellular stainings Gr1 Alexa Fluor700, CD8 FITC, CD4 APC Fire, CD11b BV570, PD-L1 BV421, NK1.1 BV605, CD19 Spark Blue (Biolegend), CD25 PerCP-eFluor710 (Thermofisher), CD83 BV750, PD-1 BV650 (BD Bioscience) and for intracellular stainings CTLA-4 PE/Cy7, CD3 PerCP, and Foxp3 Alexa Fluor 647 (Biolegend) were used. Data were analyzed using SpectroFlow™ Version 2.2.0.3. and FlowJo™ Version 10.6.1.
CircRNA circ_0001821 predicts an unfavorable prognosis and promotes the proliferation of multiple myeloma
Published in Hematology, 2021
Lin Liu, Feng Zhang, Jiajia Li
RPMI-8226 and NCI-H929 cells were seeded into 6-well plates in triplicate at a density of 3.0 × 106 cells/well, and transiently transfected with pcDNA4.0 or pcDNA4.0 + circ_0001821 and si-circ_0001821 or si-NC, respectively. 24 h after cell transfection, cell apoptosis was detected by performing AnnexinV-FITC/PI double staining flow cytometry analysis, according to the manufacturer’s instructions. Briefly, the transfected cells were washed twice with warm PBS, and resuspended with a binding buffer at a final concentration of 1 × 106 cells/ml. Then, 5 μl of AnnexinV-FITC (BD Biosciences, MA, USA) was added to the cells followed by incubation for 30 min at 37°C in dark. Before loading the samples, 2 μl of propidiumiodide (PI; BD Biosciences) was added to each tube. Cell apoptosis rate was determined with a FACSCalibur™ Flow Cytometer (BD Biosciences). The FlowJo software (FlowJo, Ashland, USA) was applied to analyze the flow cytometry data.
Low CD4⁺/CD25⁺/CD127⁻ regulatory T cell- and high INF-γ levels are associated with improved survival of neuroblastoma patients treated with long-term infusion of ch14.18/CHO combined with interleukin-2
Published in OncoImmunology, 2019
Sascha Troschke-Meurer, Nikolai Siebert, Madlen Marx, Maxi Zumpe, Karoline Ehlert, Oliver Mutschlechner, Hans Loibner, Ruth Ladenstein, Holger N. Lode
For analysis of Ab-dependent effects on effector cell counts, following effector populations were examined by flow cytometry: NK cells with a cytotoxic phenotype (CD3−/CD16+/CD56dim),23 neutrophil granulocytes (CD64+), eosinophils (FSC-A vs. SSC-A) and Tregs (CD4+/CD25+/CD127−). First, 3 ml EDTA-blood samples were incubated for 7 min with 3 ml erythrocyte lysis buffer (FACS Lysing Solution, BD Biosciences, 349202) in the dark followed by centrifugation for 5 min at RT, 300 × g. Cells were then washed once (5 min, 300 × g, RT) using wash buffer (1x PBS, 2% FCS, 0.1% NaN3, pH 7.4). After supernatant was discarded, cells were stained for 20 min at +4°C in the dark with effector cell population-specific mAb (for NK cells: mouse anti-human CD16-PE (BD Biosciences, 555407), and mouse anti-human CD56-APC (BD Biosciences, 341027); for granulocytes: mouse anti-human CD45-PerCP (BD Biosciences, 347464) and mouse anti-human CD64-PE (Biozol, LS-C204449-100); for Tregs: mouse anti-human CD3-PerCP (BD Biosciences, 340663), mouse anti-human CD4-PerCP (BD Biosciences, 345770), mouse anti-human CD25-FITC (Merck, FCMAB189F) and mouse anti-human CD127-PE (BD Biosciences, 557938) in a total volume of 100 µl wash buffer. To exclude CD64+ monocytes from the neutrophil population, monocytes were stained with CD14-FITC (DAKO, F0844). For each sample, 100,000 ungated events were estimated using FACS Calibur (BD Biosciences, Heidelberg, Germany). Data were analyzed with FlowJo V10 software (Ashland, OR, USA).