Single-cell suspensions of splenocytes were obtained from naive, infected/non-treated and infected/treated mice at 8 and 14 days p.i. Spleen cell suspensions were obtained by mechanical dissociation in PBS, then filtered in 0,70 µm strainer. 20% of each spleen was used for immunophenotyping by FACS, 80% left was dedicated to neutrophils and monocytes sorting. Red blood cells were lysed (ACK, Lonza) and an enrichment with biotinylated anti-B220 (BD Biosciences), anti-CD3 (BD Biosciences), following of anti-biotin Ab magnet-bead coupled (Miltenyi) and magnetic LS-columns (Miltenyi) was performed to remove spleen lymphocytes and increase the sorting efficacy. Cells were stained with specific markers of populations of interest (Ly6G-BD BioSciences, Ly6C-BioLegend, CD11b-BD BioSciences) and neutrophils (Ly6Ghi) and inflammatory monocytes (Ly6Chi) were sorted using the BD Biosciences FACSAria device. Sorted cells (>97-98% pure) from naive, infected/non-treated and infected/treated mice showed a viability > 90%. They were cultured in U bottom 96-well plates at a density of 4 × 106 cells/ml (1 × 106 cells/250 µl/well) in 10% FBS-containing RPMI medium. After 24 h of cell culture, cell-free supernatants were collected and stored at −20°C, to allow cytokines and chemokines protein release quantification.
Facs aria device
Manufactured by BD
Sourced in United States
The FACS Aria device is a flow cytometry instrument manufactured by BD (Becton, Dickinson and Company). Its core function is to facilitate the analysis and sorting of individual cells or particles within a heterogeneous sample. The device utilizes fluorescence-activated cell sorting (FACS) technology to rapidly and accurately identify and separate cell populations based on their unique physical and biochemical characteristics.
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Lab products found in correlation
Moflo cell sorter, by Beckman Coulter (3 mentions)
Reliaprep rna miniprep system, by Promega (2 mentions)
Ls magnetic column, by Miltenyi Biotec (1 mentions)
Anti cd3, by BD (1 mentions)
Cd11b, by BD (1 mentions)
Anti b220, by BD (1 mentions)
Trypsin edta, by Fujifilm (1 mentions)
7 aminoactinomycin d, by Beckman Coulter (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Escherichia coli 0111 b4, by Merck Group (1 mentions)
Easysep magnet, by STEMCELL (1 mentions)
Fortessa, by BD (1 mentions)
Cocktail of collagenases, by Roche (1 mentions)
Facsaria, by BD (1 mentions)
Anti ifn γ, by Thermo Fisher Scientific (1 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions)
Annexin 5 apoptosis detection kit, by BD (1 mentions)
Anti cd45, by Thermo Fisher Scientific (1 mentions)
Anti cd4, by Thermo Fisher Scientific (1 mentions)
13 protocols using facs aria device
1
Isolation and Culture of Murine Immune Cells
2
Single-cell Cloning and Expansion
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Cultured cells were dissociated with 0.05% trypsin‐EDTA (Wako, Osaka, Japan) solution and then resuspended in 2% FBS‐PBS. Dead cells were eliminated using 7‐amino‐actinomycin D (Beckman Coulter, Brea, CA) staining. Single‐cell culture analysis was then performed as previously described 14 , 24 , 25 . Individual isolated cells were sorted into 96‐well culture plates using a FACSAria device (BD Biosciences), and the wells were visualized by light microscopy 10–16 h after sorting to confirm that each well contained only one cell. After isolation of each clone, the cells were expanded and subjected to flow cytometry analysis.
3
Investigating Antigen-Specific B Cell Responses
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Fas+GL7-IgM+CD38+CD73-CD19+ cells were sorted with a FACSAria device (BD Biosciences), and then they were stained with 5 μM carboxyfluorescein succinimidyl ester (CFSE, Life Technologies) in PBS with 0.1% bovine serum albumin (BSA, Sigma-Aldrich) for 20 min at 37°C. The cells were then stimulated with Pc-iRBCs (1 B cell: 3 Pc-iRBCs) or LPS (10 μg/ml, from Escherichia coli 0111:B4, Sigma-Aldrich). Proliferation was evaluated after 72 h of culture at 37°C in a 5% CO2 atmosphere, whereas supernatants were collected at 7 days to evaluate the total and anti-parasite IgM levels.
4
Isolation of Murine Splenic CD4 T Cells
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Blood and spleen cells were washed and maintained in cold RPMI 1640 supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), 2-mercaptoethanol (50 μM), L-glutamine (2 mM), sodium pyruvate (1 mM) and 3% heat-inactivated fetal calf serum. All supplements were purchased from Life Technologies (USA). Leukocytes were obtained in 70% Percoll gradient (GE Health Care, USA). For the cell proliferation assay, spleen CD4 T cells were magnetically purified by negative selection. Non-CD4 T cells were labeled with biotinylated antibodies and streptavidin-coated magnetic particles and then were separated using an EasySep magnet (Stem Cell Technologies, Canada). For the calcium flux assay, spleen CD4 T cells were magnetically purified (LS columns) by positive selection using anti-CD4 microbeads with autoMACS (Miltenyi Biotec, Germany). For adoptive transfer, spleen CD4 T cells were magnetically purified (LS columns) by negative selection using anti-CD19, -IAb and -CD8 microbeads with autoMACS (Miltenyi Biotec, Germany) and then were sorted using a FACS Aria device (BD Biosciences, USA). In some experiments, spleen CD4 T cells were magnetically purified by negative selection using an EasySep magnet and then were sorted using a FACS Aria device.
5
Quantifying Lung ILC2 IL-13 Production
6
Comprehensive Immune Cell Profiling by Flow Cytometry
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The following antibodies were used for flow cytometry analysis: anti-IFNγ (XMG1.2, 1:50), anti-CD45.1 (A20, 1:100), anti-CD45.2 (104, 1:100), anti-IL1β-APC (NJTEN3, 1:50), anti-CD44 (IM7, 1:100), anti-CD62l (MEL-14, 1:100), anti-F4/80 (BM8, 1:100), anti-CCR7 (4B12, 1:100), anti-CD4 (RM4-5, 1:200), anti-CD8α (53-6.7, 1:200), anti-H-2Kb (AF6-88.5.5.3, 1:200), anti-Ly6G (RB6-8C5, 1:100), anti-MHC class II (I-A/I-E) (M5/114.15.2, 1:100) and anti-CD11c (N418, 1:100) were from eBioscience. Anti-CD103 (2E7, 1:100) was from Biolegend. Annexin V apoptosis Detection kit (556547) was from BD Pharmingen. For intracellular cytokine staining, Cytofix/Cytoperm plus kit (BD, cat. 55508) was used. For Foxp3 staining, Foxp3 staining buffer set (eBioscience, cat. 00-5523-00) was used. Multiple-colour flow cytometric analysis was performed using FACSAria (BD Biosciences; Franklin Lakes, NJ, USA). For FACS sorting, cells stained with FACS
antibodies were sorted on BD FACSAria device. FlowJo software was used for data acquiring and analysis.
antibodies were sorted on BD FACSAria device. FlowJo software was used for data acquiring and analysis.
7
Multicolor Flow Cytometry Analysis of Immune Cells
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For analysis of the percentages of CD45+CD4+ T cell, CD45+CD8+ T cell, CD45+CD4+CD25+ T cell, CD45+CD8+CD25+ T cell, CD45+F4/80+ cell, CD45+CD11b+ cell and CD45+MHCII+ cell, the IELs from small intestine or colon and mononuclear cells (MNCs) from LN cells obtained above were stained for tube 1: CD45 (APC Cy7, clone 30-F11), CD4 (FITC, clone RM4-5), CD8A (PE, clone 53-6.7), CD25 (APC, clone PC61) or tube 2: CD45 (APC Cy7, clone 30-F11), F4/80 (PE, clone BM8), MHCII (PerCP-CY5.5, clone M5/114.15.2), CD11b (APC, clone M1/70) antibody at room temperature for 20 min and sorted on a BD FACSAria device. All antibodies were from BD Biosciences (Franklin Lakes, NJ) except anti-F4/80 and anti-CD11b from Biolegend (San Diego, CA).
8
BATF Expression in T Cells
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Peripheral blood mononuclear cells (PBMCs) were freshly isolated from 10 mL EDTA anticoagulated venous blood using Ficoll density-gradient centrifugation and resuspended in RPMI-1640 medium (Invitrogen-Gibco, USA), supplemented with 10% heat-inactivated FBS (Invitrogen-Gibco, USA). To investigate the expression of BATF in peripheral blood T cells, PBMCs (1 × 106) were surface stained with CD4-PB (RPA-T4, BD Pharmingen), CD8-PE-Cy7 (RPA-T8, BD Pharmingen) and PD-1-FITC (MIH4, eBioscience). Cells were then fixed and permeabilized with Cytofix/Cytoperm buffer according to the manufacturer's instruction, stained with BATF-APC (687706, R&D Systems). Stained samples were fixed with 2% paraformaldehyde (PFA), and detected on a BD FACS Aria device. Data were analyzed with FlowJo software (Tree Star Inc., USA).
9
Flow Cytometric Analysis of Aldehyde Dehydrogenase and CD44
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After gentle cellular detachment from the culture plate using trypsin (0.04%)/EDTA (0.03%) solution, cells were resuspended and washed in phosphate-buffered saline, and pelleted by centrifugation. CD44 staining was performed using a monoclonal antibody and reagent from BD Biosciences (San Jose, CA, USA). Aldehyde dehydrogenase detection was performed using the Aldefluor assay (StemCell Technologies, Vancouver, BC, Canada) as follows: a total of 1 × 106 cells ml−1 was resuspended in Aldefluor buffer and incubated at 37 °C for 30 min with 2.5 μl ml−1 activated Aldefluor reagent, which includes a fluorescent-labelled substrate of ALDHs. Baseline fluorescence was established using control cells in the presence of 5 μl ml−1 of 1.5 mM dimethylaminobenzaldehyde (DEAB), an ALDH inhibitor. Cell staining and analytical procedures were performed either manually or using high-content semi-automated flow cytometry as described previously (Gasparetto et al, 2004 (link)). Flow cytometric analysis and cell sorting were performed on a FACSAria device equipped with 405 nm violet, 488 nm argon, and 633 nm HeNe lasers (Becton Dickinson, San Jose, CA, USA). Data were analysed using FlowJo software (TreeStar, Palo Alto, CA, USA).
10
Expanding CCR7+ CD8+ T cells from PBMCs
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From three healthy volunteers, PBMCs (1 x 106 cells/mL) were prepared from heparinized blood (10cc) by Ficoll–Hypaque (GE Healthcare) density-gradient centrifugation. To expand CCR7+ CD8+ T cells, isolated PBMCs were stimulated using anti-CD3, IL-15, IL-2, and retinoic acid. Cells were cultured as described previously. [11 (link)] We pooled cells for microarray as opposed to single cell RNA sequencing. CCR7+ CD8+ T cells were purified by CD8–APC (SK1, IgG1,κ; BD) and CCR7-strepavidin (3D12, IgG2a, κ). The cells were sorted using an FACS Aria device (Becton Dickinson) or a MoFlo cell sorter (Beckman Coulter) to isolate CCR7+ CD8+ and CCR7- CD8+ T cells. We extracted mRNA from CCR7+ CD8+ and CCR7- CD8+ T cells using the ReliaPrep™ RNA Miniprep Systems (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions. RNA purity and integrity were evaluated by ND-1000 Spectrophotometer (NanoDrop, Wilmington, DE, USA).
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