One experimental dataset was composed of T cell polyfunctionality and target killing capacity of HIV-specific CD8+ T cell clones analysed as previously described.[9 (link)] Briefly, T cell clones from 3 HLA B*2705 HIV-1 seropositive patients were stimulated for 6 hours with serial dilutions (10-6-10-12 M) of cognate peptide (p24 Gag KK10; residues 263–272) and analysed on a BD LSRII apparatus (BD Biosciences) for intracellular expression of IFN-γ, TNF-α, IL-2 and MIP-1β as well as surface displayed marker of recent degranulation, CD107a.
Lsrii apparatus
Manufactured by BD
Sourced in United States
The LSRII is a flow cytometry instrument designed for the analysis and sorting of cells and particles. It utilizes multiple laser sources and detectors to measure various parameters of individual cells or particles passing through a fluid stream. The LSRII is capable of simultaneously detecting and analyzing multiple fluorescent and scattered light signals, providing detailed information about the physical and biochemical characteristics of the analyzed samples.
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Lab products found in correlation
Facsdiva software, by BD (2 mentions)
Click it edu alexa fluor 647 flow cytometry assay kit, by Thermo Fisher Scientific (2 mentions)
Rnase a, by Merck Group (1 mentions)
Calibur, by BD (1 mentions)
Lsrfortesa, by BD (1 mentions)
Facsaria instrument, by BD (1 mentions)
Foxp3 transcription staining buffer set, by Thermo Fisher Scientific (1 mentions)
7 protocols using lsrii apparatus
1
Polyfunctionality and Killing Capacity of HIV-specific CD8+ T Cells
2
Cell Cycle Analysis with EdU and PI
3
Cell Cycle Analysis of PML Variants
4
Cell Cycle Analysis by FACS
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Cell cycle distribution was determined by Fluorescence Activated Cell Sorting (FACS). Fixed overnight in 70% (v/v) ethanol at −20 °C, cells were washed in PBS and resuspended in PBS containing 40 μg/ml propidium iodide and 100 μg/ml RNase A (Sigma). Cells were incubated for 30 min at 37 °C and data was acquired using a BD LSRII apparatus. For each experiment, 104 cells were analyzed.
5
Efb Modulates Neutrophil Phagocytosis
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Phagocytosis of FITC-labeled bacteria (5 × 107/ml) were mixed with human plasma for 2 min at 37°C in the presence of recombinant Efb (0.1 µM) and synthetic peptides (50 µM). Freshly isolated human neutrophils (5 × 106/ml) were added, and phagocytosis was allowed for 15 min at 37°C. Paraformaldehyde was then added to stop the reaction, and phagocytosed bacteria were analyzed by flow cytometry (LSRII apparatus; BD).
6
Cell Cycle Profiling by Flow Cytometry
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Cell cycle profile was assayed by staining with or propidium iodide (PI) or BrdU incorporation as described before.34 (link) For PI staining, control or PSTPIP2-overexpression G1ME cells were fixed, permeablized, and stained with PI (1 μg/ml). Data were acquired by Flow Cytometry and cell cycle profile was processed by FlowJo software (Tree Star Inc, Ashland, OR, USA). For BrdU incorporation, cells were labeled with bromodeoxyuridine (BrdU, 3 mg/ml) for half hour, fixed, permeablized, and stained with Alexa 647-labeled anti-BrdU antibody and 4′, 6-diamidino-2-phenylindole (DAPI, 1 μg/ml). Flow cytometry was performed on an LSRII apparatus or Calibur (BD Biosciences, San Jose, CA, USA) and data were analyzed by FlowJo software.
7
Lymphocyte Profiling by Flow Cytometry
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Phenotyping of lymphocyte populations was performed by flow cytometry after preparation of single cell suspensions from lymphoid organs and staining using antibodies listed in Table S6. Single cell suspension of thymus, spleen and lymph nodes were prepared in FACS buffer (2% FBS, 1 mM EDTA, 1% penicillin-streptomycin, in PBS) by tissue homogenization with a syringe plunger against a 40 mm cell strainer. For preparation of cell suspensions from bone marrow, femur, tibia and pelvis were flushed with FACS buffer using a 10 mL syringe and a 26 gauge-needle and then passed through 40 mm cell strainer to obtain single cell suspensions. To achieve red blood cell lysis, the cell suspensions were treated with ACK lysis buffer (0.15M NH4Cl, 10mM KHCO3, 0.1mM EDTA in H2O, pH 7.2-7.4), washed and resuspended in FACS buffer. For intracellular staining the cells were first stained with a fixable viability dye prior to surface antibody staining. After cell surface staining the cells were fixed and permeabilized using the FoxP3/Transcription staining buffer set (eBioscience) according to manufacturer's protocol followed by intracellular antibody staining. Data were collected on an LSRFortesa and/or LSRII apparatus (BD Biosciences) and were analyzed with FlowJo software version 10; cell sorting was done using a FACSAria instrument (BD Biosciences).
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