Mice were prime/boost immunized and challenged as described in sections Immunization of Mice and MHV-68 Challenge, respectively. Spleens were removed from vaccinated mice and homogenized with a syringe plunger over metal grid with cell culture medium. Erythrocytes were lysed with 3 ml TAC buffer and washed. Cells were filtered by 70 μm cell strainer, counted and stained with aqua dye (Invitrogen) followed by mouse anti-CD8 PB, anti-CD4 PerCP (eBioscience), anti-PD-1 FITC and anti-CTLA-4 APC (Invitrogen) surface antibody staining as well as anti-Eomes PECy7 and anti-T-bet PE (Invitrogen) intracellular antibody staining as described in section Intracellular Cytokine Staining (ICS).
Aqua dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
Aqua dye is a laboratory dye used for staining and visualizing aquatic samples. It is a water-soluble dye that can be used to stain various biological and chemical components within aquatic environments. The core function of Aqua dye is to provide a means for researchers and scientists to identify and analyze specific elements or organisms within water-based samples.
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Lab products found in correlation
Lsrfortessa, by BD (3 mentions)
Dnase, by Roche (3 mentions)
Cytofix fixation buffer, by BD (2 mentions)
Rat igg2b anti mouse cd16 cd32 receptor antibody, by BD (2 mentions)
Rnalater, by Thermo Fisher Scientific (2 mentions)
Propidium iodide, by Merck Group (2 mentions)
Anti pd 1 fitc, by Thermo Fisher Scientific (1 mentions)
Cd27 bv605, by BioLegend (1 mentions)
Cd8 pacificblue rpa t8, by BD (1 mentions)
Cd3 h7apc sk7, by BD (1 mentions)
Cd20 bv570, by BioLegend (1 mentions)
Pd1 bv711 eh12.2h7, by BioLegend (1 mentions)
Pharm lyse, by BD (1 mentions)
Cd11c pe cy7, by BD (1 mentions)
Cd54 apc, by BioLegend (1 mentions)
Pe ccr10, by BioLegend (1 mentions)
Ccr2 pe, by R&D Systems (1 mentions)
Hla dr percp cy5, by BD (1 mentions)
Cd3 efluor 605, by Thermo Fisher Scientific (1 mentions)
Cd103 fitc, by BioLegend (1 mentions)
13 protocols using aqua dye
1
Murine MHV-68 Immune Response Analysis
2
Multiparametric Flow Cytometry of Tonsil Cells
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The following titrated, conjugated antibodies were used for flow cytometry analysis: CD3-H7APC (SK7, BD Biosciences), CD4-BV650 (SK3, BD Biosciences), CD8-Pacific Blue (RPA-T8, BD Pharmingen), CD19-FITC (J3-119, Beckman-Coulter), CD27-BV605 (O323, Biolegend), CD45RO-Cy5PE (UCHL1, BD Pharmingen), PD-1-BV711 (EH12.2H7, Biolegend), CD57-AF594 (NK-1, Novus Bio), IgD-PE (Goat polyclonal, Southern Biotech), CD20-BV570 (2H7, Biolegend), CD38-BV786 (HIT2, Biolegend). 1-2×106 tonsil-derived cells were thawed and rested for 2h in a cell culture incubator before staining. Cells were washed with PBS, BSA (0.5%), incubated (5 min, RT) with a viability dye (Aqua-dye, Invitrogen) and surface stained with titrated amounts of antibodies for 30 min at room temperature. After washing, cells were resuspended and fixed with 1% paraformaldehyde.
3
Comprehensive Lung Immune Cell Profiling
4
Comprehensive Immunophenotyping of T Cell Subsets
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Blood was collected and processed within 4 hours maximum. An ammonium chloride-based lysing reagent (BD Pharm Lyse, BD Biosciences) was used for erythrocyte deletion of 1ml of blood. After washing, cells were suspended in PBS and stained with Aqua Dye (Invitrogen) for cell viability. Cells were washed again, suspended in staining buffer and divided in four tubes. The four different panels assessed contained some common and some specific antibodies. Common antibodies were: CD3-eFluor 605, CD4-Alexa700 (eBioscience, San Diego, CA), CCR7-Horizon PE-CF594, CD38-Brillant Violet 421, HLA-DR-PerCP-Cy5.5 and CD11c-PE-Cy7 (BD Biosciences). Specific for each panel were: 1) CCR2-PE, CCR5-APC-Cy7, CXCR6-APC (R&D Systems Inc.) and CXCR3-FITC (BioLegend); 2) CD49d (α4)-FITC, β7-APC, CCR9-PE (BD Biosciences) and CD29 (β1)–APC-Cy7 (BioLegend); 3) CD103-FITC, CD54-APC, CD49a (α1)-PE and CD29–APC-Cy7 (BioLegend); 4) CD18-APC, CLA-FITC (BD Biosciences) and CCR10-PE (BioLegend). Cells were acquired using a BD LSRFortessa SORP flow cytometer (Flow Cytometry Platform, IGTP) and analyzed with FlowJo 9.3.2 software (TreeStar). Gates were drawn based on fluorescence minus one-controls and isotypes, and CD3+ CD4- phenotype was considered CD8+ T cells.
5
Comprehensive Lung Immune Cell Profiling
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For flow cytometry staining, 2.5 × 106 lung cells were incubated for 20 min at 4 °C with a purified rat IgG2b anti–mouse CD16/CD32 receptor antibody (BD) to block Fc binding in FACS buffer. For surface staining, cells were stained with fluorochrome-conjugated antibodies against CD45 (30-F11, BV605), CD11b (M1/70, AF700), CD64 (X54-5/7.1, APC), Ly6G (1A8, BV570/BV785), CD3ε (145-2C11, FITC), Ly6C (HK1.4, BV421), CD19 (6D5, FITC), CD69 (H1.2F3, Per CP Cy5.5), MHC-II (M5/114.15.2, APC eF870), CD62L (MEL-14, BV421), CD182 (SA044G4, PE), CD11c (HL3, PE-CF594) for 25 min at 4 °C. Cells were washed with PBS and stained with fixable live-dead Aqua dye (Invitrogen) for 30 min. Cells were fixed by incubation with 100 µl 1% paraformaldehyde or Cytofix™ Fixation Buffer (BD) for 20 mins at 4 °C and stored in FACS buffer. Analysis was performed on a BD LSR Fortessa. Acquisition was set to 250,000 single, live, CD45+ cells. All antibodies were purchased from BD, BioLegend, or eBioscience. Data were analyzed with FlowJo software (Tree Star). Total cell populations were quantified as the whole lung count × (%population of CD45+ cells) × (%CD45+ cell of live cells) × (proportion of lung tissue sampled).
6
Antigen-specific T cell Induction
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Induction of antigen-specific T cells was determined using ICS. PBMCs of NHPs were collected 7 days after boost vaccination, shipped over-night at 20 °C, enriched by density gradient centrifugation, and stimulated for 6 h in presence of anti-CD28 (clone CD28.2, 10 µg/ml) and anti-CD49d (clone 9F10, 10 µg/ml) antibodies and either an overlapping peptide library covering the RABV-G protein (custom made by JPT, 10 µg/peptide/ml) or media. Golgi-Plug (BD Biosciences) was added after 1 h. Cell surface staining was performed using antibodies against CD3e (APC-Cy7, clone SP34-2), CD4 (BV650, clone OKt4) and CD8 (PE-Cy7, clone RPA-T8). Intracellular staining was performed using antibodies against IL-2 (BV421, clone MQ1-17H12), IFN-γ (FITC, B27) and Granzyme B (APC, clone GB11). Aqua Dye was used to distinguish live/dead cells (Invitrogen). Cells were acquired using a Canto II flow cytometer (BD Biosciences) and flow cytometry data were analyzed using FlowJo software (Tree Star). ICS of mouse splenocytes was performed as decribed.7 (link)
7
Fetal Small Intestine Cell Isolation
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Uninterrupted stomach to caecum sections of the fetal small intestine
were dissected in cold 1x PBS (see above). The intestine was cut into 1cm
sections and washed three times with 1mM DTT in 1x PBS for 10 minutes at
37°C to remove mucus. The epithelial layer was dissociated with three
washes of 1mM EDTA in 1x PBS for 20 minutes at 37 °C and the latter wash
was preserved in RNAlater (Ambion) at −80°C for RNAseq. The
remaining lamina propria cells were dissociated with freshly prepared 1mg/mL
Collagenase IV (Gibco) and 10mg mL−1 DNAse (Roche) in cRPMI
for 30 minutes at 37°C, in a shaking water bath at 200 rpm. Mesenteric
lymph node and spleen cells were isolated by a 30-minute digestion in
Collagenase IV media as described above and then gently pressed through a
70μm strainer. Cells were separated in a 20%-40%-80% Percoll density
gradient at 400 × g for 40 minutes: T cells were recovered at the
40–80% interface, while antigen presenting cells were recovered at the
20–40% interface. All cells were washed twice with cRPMI media. Viability
was measured with propidium iodide (Sigma Aldrich) and AQUA dye (Invitrogen)
using flow cytometry.
were dissected in cold 1x PBS (see above). The intestine was cut into 1cm
sections and washed three times with 1mM DTT in 1x PBS for 10 minutes at
37°C to remove mucus. The epithelial layer was dissociated with three
washes of 1mM EDTA in 1x PBS for 20 minutes at 37 °C and the latter wash
was preserved in RNAlater (Ambion) at −80°C for RNAseq. The
remaining lamina propria cells were dissociated with freshly prepared 1mg/mL
Collagenase IV (Gibco) and 10mg mL−1 DNAse (Roche) in cRPMI
for 30 minutes at 37°C, in a shaking water bath at 200 rpm. Mesenteric
lymph node and spleen cells were isolated by a 30-minute digestion in
Collagenase IV media as described above and then gently pressed through a
70μm strainer. Cells were separated in a 20%-40%-80% Percoll density
gradient at 400 × g for 40 minutes: T cells were recovered at the
40–80% interface, while antigen presenting cells were recovered at the
20–40% interface. All cells were washed twice with cRPMI media. Viability
was measured with propidium iodide (Sigma Aldrich) and AQUA dye (Invitrogen)
using flow cytometry.
8
Multiparametric Flow Cytometry Analysis
9
Isolation and Culture of Human Fetal Spleen APCs
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Human fetal spleen cells were isolated by a 30-min digestion with freshly prepared medium in 1 mg mL−1 collagenase IV (Gibco) and 10 mg mL−1 DNAse (Roche) in complete RPMI [RPMI medium (GIBCO) without antibiotics, 10% FBS (GIBCO), 1 mM sodium pyruvate (Life Technologies), 2 mM l -glutamine (Life Technologies), 1× nonessential amino acids (Life Technologies) and 10 mM HEPES (Life Technologies)].
Digested splenic cells were gently pressed through a 70-μm strainer, and cells were separated in a 20%:40%:80% Percoll density gradient at 400g for 40 min; APCs were recovered at the 20–40% interface. All cells were washed twice with cRPMI medium. Viability was measured with Aqua dye (Invitrogen) using flow cytometry. APCs from human fetal spleen were enriched by positive selection using Easy Step Human Biotin Isolation kit (STEMCELL Technologies) and the biotinylated human HLA-DR monoclonal antibody. Cells were seeded into 96-well plates and incubated with a multiplicity of infection (MOI) of 10 bacterial cells in antibiotic-free cRPMI for 4 h at 37°C with 5% CO2 and 4% O2 to mimic hypoxic conditions in the fetal intestine58 (link) and normalize for bacterial growth. Cells were harvested by fixation and stained for flow cytometry as described below.
Digested splenic cells were gently pressed through a 70-μm strainer, and cells were separated in a 20%:40%:80% Percoll density gradient at 400g for 40 min; APCs were recovered at the 20–40% interface. All cells were washed twice with cRPMI medium. Viability was measured with Aqua dye (Invitrogen) using flow cytometry. APCs from human fetal spleen were enriched by positive selection using Easy Step Human Biotin Isolation kit (STEMCELL Technologies) and the biotinylated human HLA-DR monoclonal antibody. Cells were seeded into 96-well plates and incubated with a multiplicity of infection (MOI) of 10 bacterial cells in antibiotic-free cRPMI for 4 h at 37°C with 5% CO2 and 4% O2 to mimic hypoxic conditions in the fetal intestine58 (link) and normalize for bacterial growth. Cells were harvested by fixation and stained for flow cytometry as described below.
10
Fetal Small Intestine Cell Isolation
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