The phenotype of pDCs was assessed with the following primary mAbs (BD Horizon): CCR7-FITC (clone 3D12), CD83-PE-Cy7 (clone HB15e), CD86-PE-Cy5 (clone 233), HLA-DR-APC-H7 (clone L243), and CD123-PerCP-Vio 700 (clone AC145). CD303-PE (clone REA693) was purchase from MACS. Cells were fixed with 4% PFA for 20 min, stained for 30 min at 4°C and washed before being subjected to FACS analysis. ZIKV infectivity was assessed with the mouse anti-pan flavivirus envelope protein mAb 4G2 (RD Biotech). A ZIKV-specific polyclonal antibody targeting the envelope protein and the non-structural protein 1 (20 (link)) or the mouse anti-dsRNA IgG2a mAb J2 (Scicons) as indicated. Cells were fixed with 4% PFA for 20 min at room temperature (RT) and permeabilized with 0.1% Triton X-100 in PBS for 4 min at RT. Fixed cells were stained overnight at 4°C using 4G2 or J2 (1:500) in PBS-BSA. Then, cells were stained 20 min at RT with secondary antibody donkey anti-mouse Alexa Fluor 488 IgG (1:1000, Invitrogen) or donkey anti-mouse Cy3 (1:500, Jackson immunoResearch) in PBS-BSA, and washed before being subjected to FACS analysis. At least, 5,000 events were acquired using Cyan cytometer (Beckman Coulter). Stained cells were analyzed using FlowJo software (Tree Star, Inc., Ashaland, OR). pDCs survival was assessed using the 7-AAD assay as previously described (21 (link)).
Cyan cytometer
Manufactured by Beckman Coulter
Sourced in France
The Cyan cytometer is a flow cytometry instrument designed for cell analysis. It provides high-performance data acquisition and analysis capabilities for various applications in life science research and clinical diagnostics. The Cyan cytometer is capable of detecting and analyzing multiple parameters of individual cells within a sample, enabling researchers to gather valuable insights about cell populations and their characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Ccf 4, by Thermo Fisher Scientific (3 mentions)
Anti cd11b apc, by BD (3 mentions)
Kaluza software, by Beckman Coulter (2 mentions)
Alexa fluor 488 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Donkey anti mouse cy3, by Jackson ImmunoResearch (1 mentions)
Anti cd45 2 percp cy5, by Thermo Fisher Scientific (1 mentions)
Anti cd45.1 pe cy7, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
Intraprep permeabilization reagent kit, by Beckman Coulter (1 mentions)
Axio observer d1, by Zeiss (1 mentions)
Fluorescein, by Thermo Fisher Scientific (1 mentions)
Dy201 05, by R&D Systems (1 mentions)
Foxp3 transcription factor staining buffer, by Thermo Fisher Scientific (1 mentions)
Fixable viability dye efluor 780, by Thermo Fisher Scientific (1 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Lsrfortessa, by BD (1 mentions)
Anti pakt s473, by Cell Signaling Technology (1 mentions)
Anti pyap s127, by Cell Signaling Technology (1 mentions)
Anti pakt t308, by Cell Signaling Technology (1 mentions)
13 protocols using cyan cytometer
1
Phenotyping and ZIKV Infection of pDCs
2
CCF-4 FRET Assay for Mtb Phagosomal Rupture
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The principle of the β-lactamase CCF-4 FRET assay is summarized in S1 Fig. . To
measure the Mtb phagosomal rupture, cells were stained during 1h at
RT, with 8 μM CCF-4 (Invitrogen) in EM buffer (120 mM NaCl, 7 mM KCl, 1.8 mM
CaCl2, 0.8 mM MgCl2, 5 mM glucose and 25 mM Hepes, pH 7.3)
complemented with 2.5 μM probenecid. Cells were then stained with
anti-CD11c-PE-Cy7, anti-CD11b-PerCp-Cy5.5 (eBiosciences) or anti-CD11b-APC (BD) mAbs
andfixed with 4% PFA overnight at 4°C. Cell mortality in the same cultures of
infected cells was determined by use of Pacific Blue Dead/Live reagent (Invitrogen),
which reacts with free amines both inside and outside of the plasma membrane,
yielding log10 1 more intense fluorescent staining of dead cells.
Anti-CD45.1-PE-Cy7 and anti-CD45.2-PerCpCy5.5 were from eBiosciences. To avoid
fluorochromes with emission signals overlapping with those of CCF-4
(λem 500–550 nm and λem 410–470
nm), APC (λem 660 nm)-, PerCp-Cy5.5 (λem 696 nm)-
or PE-Cy7 (λem 778 nm)-conjugated mAbs were chosen for concomitant
cell surface staining. Cells were analyzed in a CyAn cytometer using Summit software
(Beckman Coulter, France). At least 100,000 events per sample were acquired for
in vitro assays. For in vivo detection of CCF-4
signal in CD45 congenic mouse model, 1,000,000 events per sample have been acquired.
Data were analyzed with FlowJo software (Treestar, OR).
measure the Mtb phagosomal rupture, cells were stained during 1h at
RT, with 8 μM CCF-4 (Invitrogen) in EM buffer (120 mM NaCl, 7 mM KCl, 1.8 mM
CaCl2, 0.8 mM MgCl2, 5 mM glucose and 25 mM Hepes, pH 7.3)
complemented with 2.5 μM probenecid. Cells were then stained with
anti-CD11c-PE-Cy7, anti-CD11b-PerCp-Cy5.5 (eBiosciences) or anti-CD11b-APC (BD) mAbs
andfixed with 4% PFA overnight at 4°C. Cell mortality in the same cultures of
infected cells was determined by use of Pacific Blue Dead/Live reagent (Invitrogen),
which reacts with free amines both inside and outside of the plasma membrane,
yielding log10 1 more intense fluorescent staining of dead cells.
Anti-CD45.1-PE-Cy7 and anti-CD45.2-PerCpCy5.5 were from eBiosciences. To avoid
fluorochromes with emission signals overlapping with those of CCF-4
(λem 500–550 nm and λem 410–470
nm), APC (λem 660 nm)-, PerCp-Cy5.5 (λem 696 nm)-
or PE-Cy7 (λem 778 nm)-conjugated mAbs were chosen for concomitant
cell surface staining. Cells were analyzed in a CyAn cytometer using Summit software
(Beckman Coulter, France). At least 100,000 events per sample were acquired for
in vitro assays. For in vivo detection of CCF-4
signal in CD45 congenic mouse model, 1,000,000 events per sample have been acquired.
Data were analyzed with FlowJo software (Treestar, OR).
3
Meayamycin B and ABT-737 Cytotoxicity
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In all cases, cells were treated with 3 nM meayamycin B and 5 μM ABT-737 (alone or in combination), an equal volume of DMSO as a negative control, or 133 μM cisplatin as a positive control. Cells were treated for 5 h and stained with Annexin V-FITC (BD Pharmingen, CA) and PI (BD Pharmingen, CA) following the manufacturer's protocol. The cells were then analyzed by flow cytometry using a CyAn cytometer (Beckman Coulter, CA). Data were analyzed using Summit V4.3 software.
4
Characterization of Colon CTC Lines
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A panel of antibodies (Supplementary Table S7 ) was used to characterize the nine colon CTC lines. The IntraPrep Permeabilization Reagent kit (A07803, Beckman Coulter, Brea, USA) was employed to study the expression of intracellular proteins, whereas conjugated antibodies were directly used for membrane proteins.
Labeled tumor cells were analyzed using a Cyan cytometer (BECKMAN COULTER) and data interpreted with the Kaluza software (BECKMAN COULTER). In parallel, labeled tumor cells were also loaded on glass slides, using a cytospin, and observed under a fluorescent microscope (Axio Observer D1, ZEISS, Oberkochen, Germany).
Labeled tumor cells were analyzed using a Cyan cytometer (BECKMAN COULTER) and data interpreted with the Kaluza software (BECKMAN COULTER). In parallel, labeled tumor cells were also loaded on glass slides, using a cytospin, and observed under a fluorescent microscope (Axio Observer D1, ZEISS, Oberkochen, Germany).
5
Phagosomal Rupture in Mtb Infection
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PMA-differentiated THP-1 cells were infected with Mtb WT or Mtb Δppe-25-pe19 strains at MOI of 1. At day 3 p.i., the phagosomal rupture was assessed by Fluorescence Resonance Energy Transfer (FRET) assay as previously described [47 (link)]. Briefly, cells were stained with 8 μM CCF-4 (Cephalosporin core linking a 7-hydroxyCoumarin to a Fluorescein) (Invitrogen) in EM buffer (120 mM NaCl, 7 mM KCl, 1.8 mM, CaCl2, 0.8 mM MgCl2, 5 mM glucose and 25 mM Hepes, pH 7.3) complemented with 2.5 μM probenecid, during 1h at room temperature. Cells were then washed in PBS and stained with APC-anti-CD11b (BD Pharmingen) mAb in FACS buffer. Cells were then fixed with 4% paraformaldehyde overnight at 4°C and were analyzed in a CyAn cytometer (Beckman Coulter, France). Human IL-1β and IFN-β were quantified in the culture supernatants of these infected THP-1 cells at 24h p.i. by use of (DY201-05, R&D Systems) and (41410, PBL Assay Science) kits, respectively.
6
Multiparametric Analysis of Atherosclerotic Plaque Cells
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Cells obtained from human atherosclerotic plaques and ApoE-KO mice were analyzed following the complete panel of antibodies (Tables S1 , S2 ). Surface staining was performed by incubating the cells with selected antibodies at 4°C for 30 min in PBS. Intracellular staining of cytokines and transcription factors was performed with Foxp3 Transcription Factor Staining Buffer (eBioscience) in accordance with the manufacturer's instructions. Dead cells were excluded using Fixable Viability Dye eFluor® 780 (eBioscience). Human samples were acquired on the BD LSRFortessa cell analyzer (BD Biosciences), while mouse samples were run on the BD LSRFortessa X20 (BD Bioscience).
Regarding in vitro experiments, intracellular FOXP3 and LC3-II staining was performed using PFA 4% and 90–100% Met-OH for fixation and permeabilization, according to Cell Signaling protocol. Dead cells were excluded by staining with NIR (Live Dead Fixable Near-IR Dead Cell stain kit; Invitrogen) according to the manufacturer's instructions. Samples were run on a Cyan cytometer (Beckman Coulter).
Autophagic flux was measured by FACS analysis using antibody against LC3-II-PE (Cell Signaling clone D-11), in the presence or absence of the modulators of autophagy as described above.
Samples were analyzed with FlowJo software, version 10.5.3.
Regarding in vitro experiments, intracellular FOXP3 and LC3-II staining was performed using PFA 4% and 90–100% Met-OH for fixation and permeabilization, according to Cell Signaling protocol. Dead cells were excluded by staining with NIR (Live Dead Fixable Near-IR Dead Cell stain kit; Invitrogen) according to the manufacturer's instructions. Samples were run on a Cyan cytometer (Beckman Coulter).
Autophagic flux was measured by FACS analysis using antibody against LC3-II-PE (Cell Signaling clone D-11), in the presence or absence of the modulators of autophagy as described above.
Samples were analyzed with FlowJo software, version 10.5.3.
7
Flow Cytometric Analysis of Immune Cell Signaling
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Splenocytes from mice were resuspended in PBS, and Fc receptors were blocked in suspension with human γ-globulin at a final concentration of 0.02 µg/µL (Octapharma) for 20 min at 4ºC. The cells were then incubated with any of the following antibodies in 50 µL of PBS for 20 min at 4ºC: Pacific Blue-conjugated anti-CD19 (Biolegend, 115,523, dilution 1:100), anti-pAkt S473 (Cell Signaling, 4060S, dilution 1:200), anti-pAkt T308 (Cell Signaling, 13038S, dilution 1:200) or anti-pYAP S127 (Cell Signaling, 4911S, dilution 1:200). After being washed with PBS, the cells were incubated with an anti-rabbit TRITC-coupled secondary antibody (Jackson ImmunoResearch, 111-025-003, dilution 1:200) for 20 min at 4ºC. After being washed with PBS, the cells were fixed with paraformaldehyde (2%). Flow cytometry was performed on a CYAN cytometer (Beckman Coulter), and the data were analyzed using FlowJo software, version 10.0 (TreeStar).
8
Cell Cycle Analysis with Melphalan and GSH
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After cell culture with melphalan alone or in combination to 5 mM GSH, cell cycle analysis was performed with the APC BrdU Flow Kit (BD Biosciences, Franklin Lake, NJ, USA) according to the manufacturer protocol and an additional step of DNA staining with 4′,6′-diamidino-2-phenylindole (DAPI) (2 µg/mL). For all flow cytometry experiments, fluorescence was quantified using a Cyan cytometer and resulting data analyzed with the Kaluza software (Beckman Coulter, Fullerton, CA, USA).
9
Intracellular S100 Protein Expression
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Intracellular expression of the S100 protein in WM-266-4 and MV3 cells was determined by flow cytometry using a Cyan cytometer (Beckman-Coulter, Villepinte, France) and a fixation/permeabilization kit (Beckman Coulter, Brea, USA). The two anti-S100 antibodies (clones 8B10 and 6G1) used in the EPISPOT assay were tested to confirm S100 expression in these melanoma cell lines.
10
T Cell Activation Assay
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Fractionated CD3+ T cells were treated with different reagents as described in Results , followed by stimulation with plate-coated anti-CD3 and anti-CD28 (eBioscience, catalog no. 16-0031-85 and 16-0281-85) for 48 hr. Cells were then stained with antibodies against CD4, CD8, and surface activation markers (CD25, CD44, and CD69) (eBioscience, catalog no. 15-0041-82, 11-0081-81, 48-0253-82, 25-0691-81, and 47-0441-82, respectively). Appropriate fluorochrome-conjugated isotype matched antibodies were used as negative controls. Analysis was carried out in a Beckman Coulter Cyan cytometer, and data were analyzed using Kaluza. See Supplemental Experimental Procedures for detailed procedures.
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