All cells were stained with Live/Dead (ThermoFisher, L34966) in PBS for 30 minutes at 4°C then washed with FACS buffer. Next, the cells were stained with fluorescently-labeled antibodies against cell surface proteins (Table S1 ) in FACS buffer for 25 minutes at 4°C then washed with FACS buffer. Cells were then fixed with 2% PFA for 7-10 minutes at room temperature and washed with FACS buffer. If cells were sorted, they were not fixed. Finally, cells were re-suspended in FACS buffer and stored at 4°C until analyzed. Fixed samples were run on the Attune NxT (Thermofisher; one-week liposome uptake experiments) or CyAN ADP LX (Beckman Coulter; 4 hour and 24 hour liposome uptake and one-week macrophage subset experiments) and live cells were sorted on the INFLUX (BD).
Cyan adp lx
Manufactured by Beckman Coulter
Sourced in United States
The CyAn ADP LX is a high-performance flow cytometer designed for advanced research applications. It features a modular design with up to 10 laser excitation sources and 18 fluorescence detectors, enabling comprehensive multiparameter analysis of complex biological samples. The instrument provides accurate and reliable data acquisition, with advanced optics and electronics for enhanced sensitivity and resolution.
Automatically generated - may contain errors
Lab products found in correlation
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Fluorescein isothiocyanate (fitc), by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
123count beads, by Thermo Fisher Scientific (1 mentions)
Live dead aqua, by Thermo Fisher Scientific (1 mentions)
B220 apc cy7 ra3 6b2, by BD (1 mentions)
Cd45 apc, by Thermo Fisher Scientific (1 mentions)
Cd4 fitc rm4 5, by BD (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
7 protocols using cyan adp lx
1
Multiparameter Flow Cytometry Analysis
2
Quantifying Met Surface Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To observe Met expression on cellular surface, ∼2 × 105 cells, resuspended in 100 μl of PBS added with 1% FBS, were stained 20 min at RT, in the dark, with DN30 and DO24 mAbs previously conjugated with PE-Cy7 fluorochrome (using Lightning-Link PE-Cy7 Tandem Conjugation Kit, Innova Biosciences, Cambridge, UK). After wash, samples were analysed on a CyAn ADP LX nine-color analyser (Beckman Coulter, Brea, CA, USA). Dead cells were eliminated by excluding DAPI-positive population.
3
Multicolor Flow Cytometry Immunophenotyping
4
Characterizing SPATE Binding to Leukocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine binding of SPATEs to different leukocyte subpopulations, SPATEs were labeled with FITC (Pierce Biotechnology). Therefore, 1 mg/mL of each SPATE were incubated with 100 µg/mL FITC in 0.1 M sodium carbonate buffer (pH 9.6) for 1 hour at room temperature. Using desalting columns from the same manufacturer, FITC-labeled SPATE (SPATE-FITC) were separated from unbound FITC. For binding of SPATEs-FITC to leukocytes, 1 million total leukocytes were incubated with increasing concentrations of SPATE-FITC in HBSS/1% BSA buffer for 30 minutes on ice. Alternatively, cells were incubated with heat-denatured SPATE labeled with FITC or a combination of equimolar concentrations of unlabeled SPATE and SPATE-FITC. SPATE binding on specific cell types was identified through “forward and side scatter” gates plus cell type-specific lineage markers including anti-CD3, -CD14, -CD16, -CD19, and -CD56( [12] (link)) (Figure S1 ). After washing the cells twice with HBSS/1% BSA, fluorescence was measured on a flow cytometer (Beckmann Coulter Cyan ADP LX. Brea, California) and data were analyzed with FloJo version 4.5 (TreeStar, Ashland, OR).
5
Intracellular and Membrane Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Formaldehyde-fixed cells were permeabilized with Triton X-100 (intracellular FACS) or not (membrane proteins), washed with PBS-1%BSA and incubated with antibodies listed in Supplementary Table S1 . Samples were analyzed on a CyAn ADP LX nine-color analyzer (Beckman Coulter, Brea, CA, USA).
6
Comprehensive FACS Analysis of Immune Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For FACS analysis cells were stained for 20 minutes at 4 °C by the application of the respective antibodies: aCD4 PE (RM4-5), aCD8 PE-Cyanine7 (53-6.7), aCD3 APC (OKT3), aCD14 APC (61D3), aCD31 (WM-59) (all from eBioscience; Frankfurt am Main, Germany). For live dead discrimination propidium iodide (Molecular Probes, Eugene, Oregon) was used. MHC-I staining was performed for 45 minutes at 4 °C using Strept-Tactin APC backbone (IBA GmbH, Göttingen, Germany). Data were collected by flow cytometry on a CyAn ADP Lx (Beckman Coulter) and analyzed with FlowJo software (TreeStar). Absolute cell numbers were calculated using 123 count beads (Thermo Fisher Scientific, Waltham, USA).
7
Comprehensive Lymph Node Immune Cell Analysis
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!