Neural cells were isolated from synchronized first larval stage worms as previously described [27 (link)] and filtered into sterile FACS tubes. Briefly, staining with near IR live/dead fixable dye (Invitrogen) of the isolated neural cells was done before performing FACS sorting. The BD FACSAria II sorter was used to separate the GFP+ neural cells from the non-GFP cells, and FACSDiva 6.1.1 software was used to analyze the sort (IU Flow Cytometry Core Facility). Sorted neural cells were collected into conical tubes with TRIzol (Invitrogen), snap-frozen in liquid nitrogen and stored at −80°C. For sorting transgenic animals, the COPAS BioSelect instrument (IU Flow Cytometry Core Facilty) was used to isolate GFP+ animals based on Time of Flight (TOF) and Extinction (Ext). 250 transgenic GFP+ animals were sorted per strain and collected on unseeded 10 cm NGM plates.
Live dead fixable dye
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Live/Dead Fixable Dye is a cell viability staining solution used to distinguish between live and dead cells. It is a fluorescent dye that binds to cellular proteins, providing a quantitative assessment of cell health. The dye can be detected using flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Lsrfortessa, by BD (6 mentions)
Brefeldin a, by Merck Group (5 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (5 mentions)
Lsrii flow cytometer, by BD (5 mentions)
Ionomycin, by Merck Group (4 mentions)
Facsaria 2 sorter, by BD (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Cytofix cytoperm, by BD (3 mentions)
Lsrfortessa flow cytometer, by BD (3 mentions)
Lsrii cytometer, by BD (3 mentions)
Fc block, by BD (2 mentions)
Lsrfortessa x 20 cytofluorimeter, by BD (2 mentions)
Cytofix, by BD (2 mentions)
Cytofix cytoperm kit, by BD (2 mentions)
Fortessa, by BD (2 mentions)
Ifn γ, by Thermo Fisher Scientific (2 mentions)
Golgistop, by BD (2 mentions)
Cyan adp, by Beckman Coulter (2 mentions)
Streptavidin microbeads, by Miltenyi Biotec (2 mentions)
56 protocols using live dead fixable dye
1
Isolation and FACS Sorting of Neural Cells
2
Detailed Characterization of Thymocyte Populations
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Single cell suspension of spleen and thymus from WT and CD3e KI mice were prepared in RBC lysis buffer (Biolegend) using a gentle MACS tissue dissociator (Miltenyi Biotech) and resuspended at 1 × 107 cells/ml in PBS (Corning) containing 2% HI-FBS (VWR), and 1 mM EDTA (Corning). 1 × 106 cells were pelleted in 96 well round bottom plates and resuspended in PBS + 2%FBS + 1 mM EDTA (Flow buffer) containing Fc-block (BD), and live/dead fixable dye (Invitrogen) for 20 min. Cells were re-pelleted and resuspended in surface staining cocktail containing antibodies against mouse TCR-beta, CD8, CD4, B220, CD25, CD5, CD44, CD69 at concentrations recommended by manufacturer. Cells were washed 2 times with flow buffer and fixed with cytofix (BD) followed by 2 washes according to manufacturer’s instructions. Cells were acquired on an LSRFortessa X-20 (BD), and analyzed using Flowjo V10.6.2. Thymocytes were analyzed from the live B220-negative population, using surface CD8 and CD8 to define SP, DP, and DN thymocytes. Surface CD44 and CD25 on DN thymocytes was used to define the DN1 (CD44-pos CD25-neg), DN2 (CD44-pos, CD25-pos), DN3 (CD44-neg, CD25-pos) and DN4 (CD44-neg, CD25-neg) transition stages. Positively selected DP and SP thymocytes were identified by surface CD5 and CD69 upregulation. All populations were quantified as a relative percentage of the parent population.
3
Isolation and Sorting of Neural Cells
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Neural cells were isolated from adult worms as previously described (for detailed protocol, see Supplemental Material ; Kaletsky et al. 2018 (link)) and filtered into sterile FACS tubes. Filtered cells were stained with near IR live/dead fixable dye (Invitrogen) before FACS sorting. The BD FACSAria II sorter was used to sort GFP expressing neural cells from the non-GFP population, and FACSDiva 6.1.1 software was used for analysis (Flow Cytometry Core Facility). Cells were sorted into TRIzol (Invitrogen), snap-frozen, and stored at −80°C.
4
Fluorescent Labeling and Analysis of Anti-PD-1 Antibody
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For labeling anti-PD-1, dialysis of antibody (1 mg/ml) was done into 50 mM bicarbonate buffer, pH 9.0 for two times followed by incubating the antibody with DY-490 (5:1 molar ratio) in the dark for 1–2 h. The labeled Ab was then dialyzed in PBS at 4 °C at least three times. DY-490 labeled anti-PD-1 mAb was later administered in mice for flow cytometry studies and microdistribution studies using fluorescence microscopy. For T-cell exhaustion analysis, tumor cells prepared as described below were stained with live/dead fixable dye (Invitrogen; CAT#: L34857, 500 × diluted), Fc-block (BD, CAT#: 553142, 100 × diluted), and the antibody against CD16/CD32 (100 × diluted), CD45-AF700 (Biolegend, CAT#: 103127, 200 × diluted), APC-CD8a antibody (Biolegend, CAT#: 100712, 200 ×diluted), and PD-1-FITC (Biolegend, CAT#: 135213, 200 × diluted). For detecting labeled PD-1 antibody binding to CD8+ T cells, cells were stained with live/dead fixable dye, Fc-block, and the antibody against CD45 (200 × diluted) and CD8a (200 × diluted). Cells were incubated with the mixture of mentioned antibodies for 30 min at 4 °C and then washed twice before the flow cytometry analysis.
5
SARS-CoV-2 Spike Protein Expression
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HeLa cells were plated at 5 × 105 cells/well in 6-well plates 2 days before infection to allow adhesion and then infected with GRAd vectors at MOI of 150 for 48 h. Cells were then harvested by pipetting with cold PBS + 5 mM EDTA and aliquoted in 5 mL polystyrene FACS tubes (BD Biosciences, San Jose, CA, USA). Cells were then stained with Live/Dead fixable dye (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA); recombinant human His-tagged ACE2 protein (RayBiotech, Peachtree Corners, GA, USA) was incubated on cells before adding any antibodies and then detected with anti-His antibody (Sigma, Thermo Fisher Scientific, St. Louis, MO, USA). Spike protein expression was detected with anti-SARS-CoV-2 Spike protein S-2 subunit mouse monoclonal antibody (mAb) unconjugated (GeneTex, Irvine, CA, USA). Antibody binding was detected with goat anti-mouse IgG mAb conjugated with Alexa Fluor 647 fluorochrome (Sigma). Sample acquisition was performed on a LSRFortessa X-20 cytofluorimeter (BD Biosciences), and sample analysis was performed with FlowJo (Tree Star, Ashland, OR, USA).
6
Characterization of Immune Cell Profiles in Chagas Disease
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This experiment included 17 healthy individuals, 7 asymptomatic patients with positive serology for Chagas disease and 9 with Chagas heart disease. Cells from all experimental groups were cultured for 16–20 h after treatment.
PBMC were stained with LIVE/DEAD™ fixable dye (Invitrogen) at room temperature for 15 min and labeled with the following antibodies at 4°C for 30 min: CD14 (#E-AB-F1209C, Elabscience), CD16 (#E-AB-F1005M, Elabscience), HLA-DR (#E-AB-F1111H, Elabscience), and CCR2 (#357209, Elabscience). Then, cells were washed, fixed and acquired using a FACS Canto (Becton Dickinson). Post-acquisition analysis was performed using FlowJo version 10 software (FlowJo LLC, Ashland, Oregon, USA). In all cases, isotype-matched mAb were used as controls.
PBMC were stained with LIVE/DEAD™ fixable dye (Invitrogen) at room temperature for 15 min and labeled with the following antibodies at 4°C for 30 min: CD14 (#E-AB-F1209C, Elabscience), CD16 (#E-AB-F1005M, Elabscience), HLA-DR (#E-AB-F1111H, Elabscience), and CCR2 (#357209, Elabscience). Then, cells were washed, fixed and acquired using a FACS Canto (Becton Dickinson). Post-acquisition analysis was performed using FlowJo version 10 software (FlowJo LLC, Ashland, Oregon, USA). In all cases, isotype-matched mAb were used as controls.
7
Multiparametric Immune Profiling of PBMCs
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PBMCs were stained with live/dead fixable dye (Invitrogen) for 30 min at room temperature, and labeled with the following antibodies for 20 min at 4°C: CD14 (#562335, BD Biosciences), CD16 (#302018, Biolegend), CD56 (#318344, Biolegend), CD33 (#562854, BD Biosciences), CD86 (#305412, Biolegend), CD64 (#IM1604U, Beckman Coulter), CCR2 (#FAB151P, R&D Systems), HLA-DR (#4333608, Thermo Fisher Scientific). Samples were acquired on a BD Fortessa flow cytometer (BD Biosciences) and analyzed using FlowJo software (TreeStar).
8
SARS-CoV-2 Peptide-Stimulated Immune Responses
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Mouse splenocytes were used freshly isolated, while monkey PBMCs were stimulated after thawing and overnight resting in culture medium. Cells were plated at 1 × 106 per well in a 96-well round-bottom plate and stimulated with either SARS-CoV-2 peptide pool S-1 or S-2 or with DMSO (Sigma) as a negative control, all in the presence of anti-CD28 antibody (BD Pharmingen), for 18 h (mouse cells) or 5 h (monkey cells) in the presence of brefeldin A (Sigma). Cells were then washed to stop the stimulation, stained with Live/Dead fixable dye (Invitrogen), fixed and permeabilized with BD Cytofix/Cytoperm Kit (BD Biosciences) following the manufacturer’s instructions, and then stained with specific fluorochrome-conjugated antibodies. Sample acquisition was performed with the LSRFortessa X-20 cytofluorimeter (BD Biosciences), and sample analysis was performed with FlowJo (Tree Star). Mouse antibodies included the following: CD3 Alexa Fluor 647, CD4 BV421, CD8 BUV395, IL-17a PerCP-Cy 5.5, IFN-γ BV650, IL-2 APC-R700 (BD Biosciences), IL-4 phycoerythrin (PE), and IL-13 PE (Invitrogen); NHP antibodies included the following: CD3 AF700, CD4 PerCP-Cy 5.5, CD8 PE (BD Biosciences), and IFN-γ fluorescein isothiocyanate (FITC; U-CyTech).
9
Isolation and Analysis of Gut Lymphocytes
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Resected jejunum gut tissue was obtained from patients undergoing elective abdominal surgery. All patients signed a release to allow the unrestricted use of discarded tissues for research purposes and all protected patient information was de-identified to the laboratory investigators. Tissues were opened longitudinally, and trimmed of excess fat, the mucosal layer and extraneous material, such as stitched portions, damaged tissues or connective tissues. Gut tissue was then washed with PBS supplemented with Streptomycin, Penicillin, and Amphotericin B and incubated at 37 °C with PBS containing 1 mM EDTA for 2 cycles of 45 min to remove epithelial cells. The lamina propria layers were cut into 1 g pieces and incubated while shaking with AIMS V media containing antibiotics, 0.5 mg collagenase D and 10 μg DNase I for 3 cycles of 45 min at 37 °C. The digested tissues were collected and washed with PBS and resuspended in RPMI1650 containing 10% human serum. For analysis of T cell activation, isolated lymphocytes were labelled with the LIVE/DEAD fixable dye (Invitrogen) to exclude dead cells from the analysis. The cells were washed with staining buffer, then surface staining was performed as above with Pe-labelled anti-CD45. Cells were washed 2 times, enumerated with a LSR II (BD Biosciences) and analyzed using FlowJo software (Treestar).
10
Monoclonal Antibody Staining for Flow Cytometry
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Monoclonal antibodies for flow cytometric analysis were obtained from BD Biosciences (San Jose, CA, USA), Biolegend (San Diego, CA, USA) or eBioscience (San Diego, CA, USA) and carefully titrated prior to use. Cells were first stained with Live/Dead fixable dye (Invitrogen, Grand Island, NY, USA) before surface and/or intracellular staining. Staining of surface antigen was performed in fluorescence-activated cell sorting buffer for 10 min at 4 °C in the dark. All flow cytometric analysis was performed with a BD Fortessa (Becton, Dickinson, Mountain View, CA, USA) or Galios (Beckman Coulter) instrument; and data was analysed with the FlowJo 9.6 software (Ashland, OR, USA).
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