HLA-B*51:01–TI8 tetramers were generated as previously described (7 (link)). CTL clones were stained with the tetramers, anti-CD8 mAb, and 7-aminoactinomycin D (7-AAD), and then tetramer+CD8+7-AAD− cells were sorted by using an FACSAria I (BD Biosciences). The cryopreserved PBMCs from four chronically HIV-1–infected HLA-B*51:01+ individuals were stained with B51-TI8 tetramers, anti-CD8 mAb, and 7-AAD. HLA-B*51:01–TI8 tetramer+ CD8+ 7-AAD− cells were sorted into 96-well plates (Bio-Rad) by using an FACSAria I (BD Biosciences). For samples from sorted single HLA-B*51:01–TI8-specific CD8+ T cells and CTL clones, unbiased identification of TCR αβ-chain usage was assessed as previously described (8 (link)). A modification to the protocol was applied by using illustra ExoStar (GE Healthcare), which contains alkaline phosphatase and exonuclease 1 to remove unincorporated primers and nucleotides from the amplification reaction prior to subsequent steps. For bulk-sorted HLA-B*51:01–TI8-specific CD8+ T cells, TCR genes were cloned with a Zero Blunt TOPO PCR cloning kit (Invitrogen), and then several clones were sequenced. Sequencing was done with a BigDye Terminator v3.1. cycle sequencing kit (Applied Biosystems) and analyzed by ABI 3500 and 3500xL Genetic Analyzer (Applied Biosystems, Carlsbad, CA). All TCR genes are identified using the ImMunoGeneTics database (http://www.imgt.org ).
Facsaria 1
Manufactured by BD
Sourced in United States, Germany, United Kingdom, Japan
The FACSAria I is a cell sorter designed for high-performance flow cytometry analysis. It provides efficient cell sorting capabilities for a wide range of applications.
Automatically generated - may contain errors
Lab products found in correlation
Verapamil, by Merck Group (10 mentions)
Propidium iodide, by Merck Group (9 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (8 mentions)
Opti mem, by Thermo Fisher Scientific (6 mentions)
Facsaria 2, by BD (6 mentions)
Facsaria 3, by BD (6 mentions)
Facscanto 2, by BD (6 mentions)
Facsdiva, by BD (6 mentions)
Hoechst 33342, by Merck Group (5 mentions)
Facsdiva software, by BD (4 mentions)
Facsaria fusion, by BD (4 mentions)
7 aad, by BioLegend (4 mentions)
Cellquest software, by BD (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
7 aminoactinomycin d, by BD (3 mentions)
Facscalibur, by BD (3 mentions)
Facsdiva 6, by BD (3 mentions)
Cd14 fitc, by Thermo Fisher Scientific (2 mentions)
Cd45 fitc, by BioLegend (2 mentions)
Cd90 pe, by BioLegend (2 mentions)
189 protocols using facsaria 1
1
Identification of TCR usage in HLA-B*51:01-restricted CD8+ T cells
2
Immune Exhaustion and Senescence Analysis
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The expression of immune exhaustion (PD-1) and senescence (CD57) markers was assessed as described previously (26 (link)). Briefly, after revival and resting overnight, PBMCs were resuspended in 1 ml PBS and then stained with violet amine reactive dye (Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature for differentiation of dead and live cells. The samples having more than 90% viability were considered for further analysis. The cells were washed again and incubated with a cocktail of antibodies [anti-Vα24 PE, anti-Vβ11 FITC (Beckman Coulter, Marseilles, France), anti-CD57 APC (Biolegend, USA), anti-CD3 PETR (Invitrogen, Carlsbad, CA, USA), anti-CD3 APC, and anti-PD1 PerCpCy5.5 (BD Biosciences)] for 30 min at room temperature. After washing, the cells were fixed in 3% paraformaldehyde, acquired on FACSAria-I (BD Biosciences, USA) and analyzed using FACSAria-I (BD Biosciences, USA) and analyzed using FACSDiva software version 6.1.3 (BD Biosciences, USA).
3
Isolation and FACS Analysis of LSK Cells
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For LSK (Lin−sca-1+c-kit+) cells soring, lineage cells are plotted as the population of B220+ (eBioscience), CD3+ (eBioscience), and Myeloid (Gr-1+Mac-1+) (eBioscience) cells in bone marrow mononuclear cells by FACS analysis using a BD FACS Aria I (BD Bioscience). After lineage depletion, lineage-negative cells were stained with anti-sca-1 (eBioscience) and anti-c-kit (eBioscience) and subjected to FACS analysis to sort the population of LSK cells (gated as Lin−/sca-1+/c-kit+) using a BD FACS Aria I (BD Bioscience). Lymphocytes from bone marrow were plotted as the population of CD4+CD8+ (BD Bioscience).
4
Enriching Glioma-Initiating Cells
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Two days after the transduction with shRNA expression retrovirus vectors (pSSCG) or shRNA/cDNA dual expression lentivirus vectors (pLE-IG), GICs were dissociated with TrypLE express (Thermo Fisher Scientific) and GFP (+)/7-AAD (−) cells were sorted by FACS ARIA I or ARIA II (Becton Dickinson) at a density of 1 cell per well into ultra-low attachment 96-well plates (Corning) in 100 μl of DMEM/F12 serum-free medium. After 2 weeks, the percentage of wells containing spheres was calculated.
5
Flow Cytometry Cell Sorting Protocol
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For cell sorting by flow cytometry, whole blood or bone marrow from two uninfected animals were first lysed using NH4Cl, then, FcRs were blocked using cynomolgus macaque serum. Cells were counted and incubated for 30 min with the following antibodies: CD11b (ICRF44), CD45 (D058-1283), CDw125 (REA705), CD123 (7G3), CD3 (REA994), CD20 (LT20), CD8a (BW135/80), CD16 (REA423), CD10 (HI10a), CD14 (TUK4) CD32a (IV.3), and CD66 (TET2). Cell sorting was performed using a FACSAria I flow cytometer (Becton Dickinson). The sorted population was smeared with a cytocentrifuge (Cytospin 2, Thermo Scientific) and then stained using May-Grünwald Giemsa. Images were acquired using a Nikon Eclipse 80i with Dxm 1200C digital camera at 60x magnification. Cells were identified by morphological criteria by a cytologist. Promyelocytes and myelocytes were considered to be pre-neutrophils (PreN), metamyelocytes and band cells immature neutrophils, and segmented neutrophils mature cells.
6
Side Population Identification Assay
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The cell suspensions were labeled (Hoechst 33342, Molecular probes – #H-3570) and dye for side population analysis. Cells were resuspended at 1 × pre-warmed (OptiMEM, Gibco, USA) containing 2% fetal bovine serum (FBS) (Gibco, USA) at a density of 106/mL. Hoechst 33342 dye was added at a final concentration of 5 mg/mL in the presence or absence of verapamil (50 mmol/L; Sigma) and the cells were incubated at 37°C for 90 min with intermittent shaking. At the end of the incubation, the cells were washed with OptiMEM containing 2% FBS and centrifuged down at 4°C, and resuspended in ice-cold OptiMEM containing 2% FBS and 10 mmol/L HEPES. Propidium iodide (Sigma, USA) at a final concentration of 2 mg/mL was added to the cells to gate viable cells. The cells were filtered through a 40-lm cell strainer to obtain single cell suspension before sorting. Analysis and sorting were done (FACS AriaI, Becton Dickinson). The Hoechst 33342 dye was excited at 355 nm and its dual-wavelength emission at blue and red region was plotted to get the side population (SP) scatter.
7
Measuring Intracellular Reactive Oxygen Species
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Cells were incubated with dichloro‐dihydro‐fluorescein diacetate (DCFH‐DA; Beyotime, Shanghai, China) at 37°C for 20 minutes. DCFH‐DA diffuses passively into cells, where it is deacetylated by esterases to form non‐fluorescent 2′,7′‐dichlorofluorescein (DCFH). The amount of fluorescence emitted correlates with the quantity of ROS in the cell. Data were acquired on a FACS Aria I (Becton Dickinson) and analyzed using FlowJo software.
8
Sorting and Isolation of Endothelial Cells and Pericytes
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Freshly isolated SVF cells were resuspended in EGM-2 medium, filtered through a 40 µm cell strainer (Greiner, Austria), and cell yield was determined. The 5 × 106 cells were incubated with 25 µL of primary antibodies anti-CD31-FITC (BD Biosciences, Austria), anti-CD146-PerCP (R&D, Germany) and anti-CD45-PE (BD), for 30 min at 4 °C in 250 µL of FACS Buffer (PBS, 0.1% BSA, 0.2 mM Glutamine) or remained unstained to serve as the control. Afterwards, the cells were washed with FACS Buffer, centrifuged at 300× g for 5 min, resuspended in 2.5 mL of FACS Buffer and filtered through a 40 µm cell strainer. Cell sorting was performed on a FACS Aria I (Becton Dickinson, Heidelberg, Germany) using a 100 µm nozzle to yield two distinct cell populations, which were as follows: endothelial cells sorted as CD31+, further referred to as ‘EC’ and pericytes sorted as CD45−/CD31−/CD146+, further referred to as ‘PC’. Unstained cells were used to determine the background autofluorescence. Cell aggregates were excluded in an FSC-W versus FSC-A gate. Sorting was performed in ‘Purity’ mode and approximately 3 × 105 EC and PC were collected in 1 mL of EGM-2. After centrifugation at 300× g for 5 min, the cell pellets were resuspended in 800 µL of TRI Reagent® (Sigma-Aldrich). Until RNA isolation, the samples were stored at −80 °C.
9
ECFC and MSC Immunophenotyping Protocol
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ECFC single-cell suspension was generated by detaching cells with TrypLE™ Express Enzyme (Gibco, USA) and resuspended to a concentration of 1 × 107 cells/ml. Samples were incubated, respectively, with anti-human CD31-FITC (eBioscience, USA), VEGFR2/KDR-PE (R&D, USA), CD144-FITC (Abcam, UK), CD34-PE (Biolegend, USA), CD45-FITC (Biolegend, USA), and CD14-FITC (eBioscience, USA). MSCs were resuspended to a concentration of 1 × 106 cells/ml and incubated, respectively, with anti-human CD29-PE (Biolegend, USA), CD90-PE (Biolegend, USA), CD14-FITC (Biolegend, USA), CD19-PE (Biolegend, USA), CD73-FITC (Biolegend, USA), CD105-FITC (Biolegend, USA), HLA-DR-PE (Biolegend, USA), CD34-PE (Biolegend, USA), CD45-FITC (Biolegend, USA), and CD31-FITC (eBiosciences, USA). 5 μl antibody solution was added into 100 μl cell suspension and incubated for 30 minutes at 4°C in the dark; 400 μl of PBS was added and cells were analyzed with FACSAria I (Becton Dickinson, USA) or Accuri C6 (Becton Dickinson, USA) and Becton Dickinson CELLQuest software.
10
Erythroid Precursor Isolation and Analysis
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Spleens were smashed and washed and the four leg bones flushed with 5 mL PBS containing 1mM EDTA and 2% fetal calf serum. Cells were individualized, filtered through a 40 μm cell strainer (BD Falcon) and incubated with 0.5 μg anti-CD16/32 antibody in 50 μL.
PBS for 30min on ice. BM cells were stained with 0.3 μg CD44-PE-Cy7 (or CD71-PE-Cy7), 0.3 μg Ter119-PE or Ter119-APC-Cy7 antibodies. Spleen cells were stained with the following lineage mix: each 0.5 μg biotinylated CD3e (clone 145-2C11), CD11b (clone M1/70), CD19 (clone 1D3), B220 (clone RA3-6B2) and Gr-1 (clone RB6-8C5) antibodies. Cells were washed and stained with Streptavidin-Pacific Blue (Thermo Fisher) and 0.3 μg CD71-PE-Cy7 and 0.3 μg Ter119-PE (or Ter119-APC-Cy7) antibodies. Lineage-negative cells were gated and plotted as CD71-PE-Cy7-versus Ter119-APC-Cy7-positive cells. Cells were analysed on a FACSAriaI (Becton Dickinson).
PBS for 30min on ice. BM cells were stained with 0.3 μg CD44-PE-Cy7 (or CD71-PE-Cy7), 0.3 μg Ter119-PE or Ter119-APC-Cy7 antibodies. Spleen cells were stained with the following lineage mix: each 0.5 μg biotinylated CD3e (clone 145-2C11), CD11b (clone M1/70), CD19 (clone 1D3), B220 (clone RA3-6B2) and Gr-1 (clone RB6-8C5) antibodies. Cells were washed and stained with Streptavidin-Pacific Blue (Thermo Fisher) and 0.3 μg CD71-PE-Cy7 and 0.3 μg Ter119-PE (or Ter119-APC-Cy7) antibodies. Lineage-negative cells were gated and plotted as CD71-PE-Cy7-versus Ter119-APC-Cy7-positive cells. Cells were analysed on a FACSAriaI (Becton Dickinson).
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