Tumors were removed from deeply anesthetized animals, digested and prepared for flow using the Tumor Dissociation Kit, mouse (Miltenyi Biotec, Bergisch Gladbach, Germany) per manufacturers protocol. In brief, tumors are cut into small pieces with scissors and placed into C-tubes (Miltenyi Biotec). Enzymes are added and the tissue further minced with the gentleMACS dissociator (Miltenyi Biotec). The samples were then incubated at 37 °C for 40 min on a rotator, and afterwards minced in the dissociator. The samples were run through a 70 μm filter and washed with Dulbecco's PBS (DPBS) without magnesium or calcium +0.5% bovine serum albumin (BSA) and 2 mM ethylenediaminetetraacetic acid (EDTA). 4′,6-diamidino-2-phenylindole (Dapi) was added as a live/dead cell marker. Flow cytometry was done on a BD LSRFortessa cell analyser (Becton Dickinson (BD), Franklin Lakes, NJ), and analysis was performed using FlowJo version 10 (FlowJo, Ashland, OR).
Lsrfortessa cell analyser
Manufactured by BD
Sourced in United States, United Kingdom, Germany
The BD LSRFortessa cell analyzer is a compact and versatile flow cytometry instrument designed for a wide range of applications. It features a flexible configuration with up to 18 parameters, enabling simultaneous detection and analysis of multiple cell populations. The instrument utilizes advanced optics and fluidics to provide high-quality data, supporting researchers in their efforts to gain deeper insights into cellular biology and function.
Automatically generated - may contain errors
Lab products found in correlation
Facsdiva software, by BD (18 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (4 mentions)
Cyan adp flow cytometer, by Agilent Technologies (3 mentions)
Propidium iodide, by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Sytox blue dead cell stain, by Thermo Fisher Scientific (2 mentions)
Cd206, by BioLegend (2 mentions)
Apc mouse lineage antibody cocktail, by BD (2 mentions)
Anti cd127 fitc, by BD (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
7 aad, by BioLegend (2 mentions)
Dulbecco s phosphate buffered saline, by Thermo Fisher Scientific (2 mentions)
Fcr blocking reagent, by Miltenyi Biotec (2 mentions)
Pi plc, by Merck Group (2 mentions)
Facs sortware sorter software, by BD (2 mentions)
Mouse fcr blocking reagent, by Miltenyi Biotec (2 mentions)
Facsdiva 8, by BD (2 mentions)
Streptavidin apc, by BD (2 mentions)
Facsdiva acquisition software, by BD (2 mentions)
Facsmelody cell sorter, by BD (2 mentions)
130 protocols using lsrfortessa cell analyser
1
Tumor Dissociation and Flow Cytometry Protocol
2
Multicolor Flow Cytometry Phenotyping
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Antibodies against the following antigens were used: anti-mouse B220 (clone 6B2), CD38 (clone 90), IgM (clone 11–41), CD24 (clone M1/69), CD21/35 (clone 7E9), CD23 (clone B3B4), and IgD (clone 11-26c) from BD Biosciences. Anti-mouse CD5 (clone 53-7.3) and CD11b (clone M1/70) from Biolegend. Anti-mouse CD11a (integrin αL, clone M17/4), CD18 (integrin β2, clone M18/2), CD29 (integrin β1, clone HMb1-1), and CD49d (integrin α4, clone R1-2) from eBioscience. Murine cells were resuspended in DPBS + 3% FCS and stained for 30 min at 4°C with different anti-mouse antibodies in the presence of anti-mouse TruStain fcX (Biolegend). Control samples were labeled with matched isotype control antibodies. The samples were resuspended in Sytox Blue Dead Cell Stain (1:1,000 in DPBS; Invitrogen) before acquisition. Samples were acquired by flow cytometry (BD LSRFortessa cell analyser, Becton Dickinson) with FACSDiva software v6.2 (Becton and Dickinson) and data were analyzed using FlowJo (Tree Star) software.
3
Flow Cytometric Analysis of Apoptosis
4
Cytometric Analysis of Immune Cells
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Splenocytes and bone marrow cells from infected mice were analysed by flow cytometry. Cells were stained with PE-conjugated anti-DNGR-1, APC-conjugated anti-CD11c, FITC-conjugated anti-MHCII and CD4, Pacific blue-conjugated anti-CD8, and Percp-conjugated anti-CD3 (all obtained from eBioscience), as previously described13 (link). For intracellular staining, cells were stimulated with BMDC pre-incubated with fixed parasite. 2 hours later, Brefeldin A was added and cells were incubated for further 4 hours. After fixation, cells were permeabilised and stained with APC-conjugated anti-INFγ. Flow cytometric analysis was performed with a BD LSRFortessa™ cell analyser (Becton Dickinson). 5.105 cells per sample were acquired and analysed with the FACSDiva or with the flowjo software.
5
Cell Cycle Analysis with EdU Labeling
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Cells were pulse-labelled with 20 μM 5-ethynyl uridine (EdU) for 45 min before cell harvest. EdU staining was performed using the Click-iT EdU kit (Invitrogen, C10424) according to the manufacturer's protocol. 4,6-Diamidino-2-phenylindole or propidium iodide (PI) were used for DNA staining. BD LSRFortessa cell analyser with the FACSDiva software was used for cell cycle analysis.
6
Cell Cycle Analysis by Flow Cytometry
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siRNA-treated cells were harvested, washed with cold PBS, and fixed by adding ice-cold 70% ethanol dropwise while vortexing. After incubation on ice for 30 min, cells were washed with PBS and treated with 50 μg/ml RNase A for 30 min at 37°C. Finally, cells were stained with 20 µg/ml PI on ice for 30 min. Flow cytometry analysis was performed on a BD LSRFortessa cell analyser. Data were analysed with Flow Jo software using Cell Cycle Analysis Tool.
7
Cell Culture and Flow Cytometry
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Experiments were performed according to [31] (link). Briefly, cells were cultured in liquid SCD media (180 rpm, 30 °C) with aliquots taken every 2–3 days for flow cytometry analysis (501 nm excitation, 586 nm/15 nm emission detection on Becton Dickinson (BD) LSRFortessa™ cell analyser, BD FACSDiva 8.0.1 software) after propidium iodide (PI) staining.
8
Multiparameter Flow Cytometry for Cell Analysis
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Multiparameter flow cytometry (MFC) and sample processing was carried out as described previously [70 (link)]. MFC analyses were performed using FC500 or Navios flow cytometers (Beckman Coulter, Miami, FL, USA). List mode files were analyzed using CXP Software version 2.0 and Kaluza version 1.0 (Beckman Coulter, Brea, CA, USA). Diagnoses were assigned according to EGIL and WHO classifications [10 ,71 (link)]. Single cell suspensions of transduced c-Kit+ cells were prepared as described elsewehere [15 (link)]. Dead cells were excluded by gating on 7AAD (Miltenyi Biotec)-negative cells. Flow cytometry analysis were performed on an LSR Fortessa cell analyser (BD Biosciences, San Jose, CA, USA) and data were analysed with FlowJo software v 10 (BD, Franklin Lakes, NJ, USA).
9
Flow Cytometry Analysis Workflow
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Fluorescence activated cell sorting (FACS) readings were acquired on LSR Fortessa cell analyser (BD Biosciences) and analysed with Flowjo software (Treestar Flowjo, Version 10.0.6). Experiments were performed in triplicate and repeated independently at least 3 times.
10
Purification and Identification of LSK Cells
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Lin− cells were purified from C57BL/6J and FA-A FVB/NJ mice by cell sorting using lineage-specific antibodies phycoerytrin conjugated (BD Pharmingen) (anti-B220 (CD45R), anti-Mac-1 (CD11b), anti-Gr1 (Ly6G/C), anti-CD3-ε, and anti-Tert-119 antibodies) at a concentration of 0.06 μg/mL and 0.02 μg/mL, respectively. For the identification of LSK cells (Lineage negative, Sca-1+, c-Kit+), Lin− cells were stained with the described cocktail, and with 0.06 μg/mL of anti-Sca-1-APC-Cy7 (BioLegend) and 0.15 μg/mL of anti-c-Kit-A647 antibodies (Southern). 4′,6-Diamidino-2-phenylindole (DAPI; Roche)-negative staining was used as a marker of cell viability. Analyses were performed in the LSR Fortessa cell analyser (BD). Transfection efficiency was also determined by FACS analysis 48 h post nucleofection. Off-line analyses were conducted with the FlowJo Software v7.6.5 (© FlowJo, LLC).
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