Cell surface and intracellular staining were performed using antibodies including CD3 (eFluor 450; eBioscience, Waltham, MA, United States; clone UCHT1), CD8a (FITC, eBioscience, clone RPA-T8; or APC-eFluor 780, eBioscience, clone RPA-T8), TRBV4-1 (PE; Miltenyi Biotec, Bergisch Gladbach, Germany; clone REA871), Ghost Dye (Violet 510; Tonbo Biosciences, San Diego, CA, United States), IFN-γ (PE-Cyanine7, eBioscience, clone 4S.B3), T-bet (BV421; BD Biosciences, San Jose, CA, United States; clone O4-46), Granzyme A (PE-Cyanine7, eBioscience, clone CB9), Granzyme B (FITC, BD eBioscience, clone GB11), Granzyme K (eFluor 660, eBioscience, clone G3H69), PRF1 (BV421, BD Biosciences, clone δG9), CCL4 (PerCP-eFluor 710, eBioscience, clone FL34Z3L), CCL5 (eFluor 660, eBioscience, clone VL1), and CXCR3 (PE-Cyanine7, eBioscience, clone CEW33D). Intracellular staining was performed on T cells stimulated with PMA (50 ng/mL; Sigma-Aldrich, St. Louis, MO, United States) and ionomycin (1 mM; Sigma-Aldrich) in the presence of GolgiStop (2/3 μL/mL, BD Biosciences) and Grid-plug (1 μL/mL, BD Biosciences) for 5 h. Flow cytometry was performed on a BD FACSCanto II flow cytometer using BD FACSDiva Software and FCS Express 5 software (De Novo Software, Los Angeles, CA, United States).
Efluor 660
Manufactured by Thermo Fisher Scientific
Sourced in United States
EFluor 660 is a fluorescent dye used in flow cytometry applications. It has an excitation maximum at 660 nm and an emission maximum at 680 nm. The dye can be used to label and detect target cells or molecules in complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Brilliant violet 421, by BioLegend (5 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Apc cy7, by BioLegend (3 mentions)
Efluor 450, by Thermo Fisher Scientific (3 mentions)
Clone bm8, by Thermo Fisher Scientific (2 mentions)
I a 1 e, by BD (2 mentions)
Ex527, by Bio-Techne (2 mentions)
Ctla 4 pe, by BD (2 mentions)
Integrin α4β7, by BioLegend (2 mentions)
F4 80, by BioLegend (2 mentions)
Apc cy7, by BD (2 mentions)
Cd103, by BioLegend (2 mentions)
Mouse anti human cd4 fitc and anti human cd25 fitc, by BD (2 mentions)
Sepmate 15 tube, by STEMCELL (2 mentions)
Facscalibur, by BD (2 mentions)
Lsm 700, by Zeiss (2 mentions)
Facscanto 2, by BD (2 mentions)
Dnase 1, by Roche (2 mentions)
Facscanto, by BD (2 mentions)
Anti cd16 cd32 mab, by BD (1 mentions)
35 protocols using efluor 660
1
Multiparametric Flow Cytometry Analysis
2
Quantification of Regulatory T Cells
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In brief, fresh heparinized whole blood samples (6 mL) were collected from patients at each time point. PBMCs were isolated using SepMate-15 tubes (STEMCELL Technologies, Vancouver, Canada) and Lymphoprep (Alere Technologies AS, Oslo, Norway), centrifuged with 1,200 g for 20 minutes in room temperature. PBMC layer at the interface was collected and twice washed at 3,500 rpm by phosphate buffer saline for 5 minutes. PBMCs were resuspended with RPMI medium-1640 (Glico technology, New Youk, NY, USA), which was used to study the number of FoxP3 Treg by flow cytometry. 1×106 cells of PBMCs were incubated with mouse anti-human CD4 FITC and anti-human CD25 FITC (BD Bioscience, San Jose, CA, USA) for extracellular staining and anti-human Foxp3 (eFluor 660, eBioscience, San Diego, CA, USA) for intracellular staining. Moreover, Isotype control was used anti-FOXP3 isotype (Rat IhG2 K, eFluor 660, eBioscience). To determine CD4+CD25+FOXP3+ Treg cells were analyzed by flow cytometry using BD FACSCalibur, BDBioscience (Fig. 1 ).
3
Multiparameter Immunofluorescence Staining
4
Multiparametric Flow Cytometry of T Cells
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Flow cytometry was performed to enumerate single-TCR and dual-TCR cells and evaluate T-cell function. Cells were incubated with Zombie Yellow (BioLegend) viability dye prior to labeling with antibodies for identification of T cells (CD4, GK1.5, APC-Cy7; CD8α, 53–6.7, PerCP-Cy5.5; Thy1.1, OX-7, AF700; Ly5.1, A20, AF700), and markers of activation (CD44. IM7, AF700; OX40, OX-86, BV711; CD103, 2E7, Pacific Blue) and exhaustion (PD-1, 29F.1A12, PE-Cy7; LAG-3, C9B7W, APC). Measurement of regulatory T cells (Tregs) and transcription factors was performed by permeabilization and fixation with True Nuclear Fix/Perm buffer and intracellular labeling for FoxP3 (150D, Pacific Blue), Bcl6 (7D1, APC), Tox (TXRX10, eFluor660, Invitrogen), Eomes (Dan11mag, eFluor660) and Nur77 (12.14, AF647). Samples were run with color and fluorescence-minus-one (FMO) controls and where applicable, cells from single-transgenic B6.TCRA-GFP and B6.TCRA-RFP mice were used as FMO controls for gating single-TCR and dual-TCR cells from B6.TCRA-GFP/RFP mice. Flow cytometry analyses were performed using FACSCanto or LSR II instruments (BD Biosciences) with FACSDiva software. Data were analyzed using FlowJo V.10 software.
5
Myosin Heavy Chain Expression in Differentiated mESCs
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Differentiated mESC cultures were trypsinized and dissociated into single cell suspensions pooling 16–48 wells of the same condition in 384-well-plates. After centrifugation, cells were fixed in 4% paraformaldehyde and incubated for 10 min at RT. Fixed cells were centrifuged and incubated with mouse anti-myosin heavy chain eFluor 660® (eBioscience, 1:100 in 0.25% saponine/2% FBS in PBS) for 1 h at RT in the dark. Following washes with 0.25% saponine/2% FBS in PBS, cells were resuspended in 500 µl 0.25% saponine/2% FBS in PBS and analyzed on a BD LSRII Aria instrument. FlowJo software was used for data analysis.
6
Multiparameter Flow Cytometry Analysis
7
Tracking CSF-Derived Antigen Uptake
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The uptake and processing of CSF-derived protein by antigen-presenting cells outside the CNS was evaluated by injecting 3 μl of DQ-OVA (2 mg/ml, Invitrogen) into the CSF of chronically infected C57BL/6 mice by i.c.m. injection. The fluorescent signal of proteolytically cleaved DQ-OVA, with a peak excitation wavelength of 505 nm and peak emission wavelength of 515 nm, was then detected by flow cytometry or confocal microscopy. For flow cytometry studies, the dural meninges, deep cervical lymph nodes, and inguinal lymph nodes were harvested and prepared as single-cell suspensions for cell surface staining 2 hr, 5 hr, or 24 hr after i.c.m. injection of DQ-OVA. For flow acquisition, fluorescence emitted by processed DQ-OVA was detected using the FL1 sensor (525/40 nm) of a Gallios flow cytometer (Beckman Coulter) or using a Cytek Aurora Flow Cytometry System. For imaging studies, mice were sacrificed 12 h after injection of DQ-OVA, and whole-mount dural meninges were fixed, then immunostained using directly-conjugated antibodies targeting MHC class II (Super Bright 436, eBioscience) and LYVE1 (eFluor 660, eBioscience). Fluorescence emitted by processed DQ-OVA was detected using 488 nm laser excitation on a TCS SP8 confocal microscope (Leica).
8
Multicolor Fluorescence Imaging of Immune Cells
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Neutrophils and neutrophil-derived EVs (nEVs) were stained with anti-mouse Ly6G antibodies (1.6 ug/mouse; PE, Brilliant Violet 421 or AlexaFluor 488 anti-Ly6G, clone 1A8, BioLegend, San Diego, CA, USA) and Kupffer cells (KCs)/Kupffer cells-derived EVs or monocytes/macrophages-derived EVs (mEVs) were visualized with PE, AlexaFluor 488 or eFluor 660 conjugated anti-F4/80 monoclonal antibodies (1.2 μg/mouse, clone BM8; eBioscience, San Diego, CA, USA). Platelets were visualized with anti-CD49b antibodies (1.2 μg/mouse; PE anti-CD49b, clone HMα2, BioLegend, San Diego, CA, USA). Endothelial cells were stained with anti-CD31 (PECAM) antibodies (5 μg/mouse; Alexa Fluor 488 anti-CD31, clone 390, BioLegend, San Diego, CA, USA). Cells and EVs were visualized at magnification of 20, 40 or 63 ×. Numbers of EVs were counted under 40× magnification, minimum 3 fields of view (FOVs) from each mouse.
9
SARS-CoV-2 Spike Protein Immune Complexes
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SARS-CoVpp-IgG immune complexes were obtained by incubating SARS-CoVpp with 30 μg/ml of either purified mouse anti-Spike or control IgG at 37°C for 1 hr. The mixture was then quickly chilled on ice for 10 min and added (100 μl/well) to ST486 cells (3×105 cells/well), which had been previously stained with 0.1% fixable viability dye eFluor 660 (eBioscience, San Diego, CA, USA). Following a 1 hr incubation on ice, cells were washed twice with cold PBS, fixed with 1% paraformaldehyde for 20 min on ice and immune complex binding was revealed by staining with 5 μg/ml FITC-conjugated goat anti-mouse F(ab’)2 (Jackson ImmunoResearch) at 4°C for 30 min. Data were collected and analysed as described above.
10
Flow Cytometry Analysis of Murine Immune Cells
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