Following incubation with labelled exosomes, BeWo cells were washed, trypsinized, washed in media containing 5 mM EDTA and finally resuspended in FACS buffer (1% FCS, 5 mM EDTA in PBS). DAPI was added prior to acquisition for gating of viable cells. Samples were measured on either: a Becton Dickinson FortessaLSR equipped with 20 mW 355 nm, 50 mW 405 nm, 50 mW 488 nm, 50 mW 561 nm, 20 mW 633 nm lasers and a ND1·0 filter in front of the FSC photodiode or a Becton Dickinson FortessaLSR equipped with 50 mW 405 nm, 50 mW 488 nm, 50 mW 561 nm, 20 mW 633 nm lasers and a ND1·0 filter in front of the FSC photodiode. Acquisition was set to record 10 000 single cells, following FSC‐A/W, SSC‐A/W gating to exclude doublets and DAPI‐negative gating to exclude dead cells. PMT voltages were adjusted after standardized CST checks minimizing the spectral overlap to increase data precision. To detect PKH‐26 fluorescence, a 561 nM laser with 582/15 nM band pass filter was used. Analysis was performed using FlowJo.
Fortessa lsr
Manufactured by BD
Sourced in United States
The Fortessa LSR is a flow cytometer designed for cell analysis and sorting. It is capable of detecting and analyzing multiple parameters of individual cells or particles within a sample. The core function of the Fortessa LSR is to provide accurate and reliable data on cellular characteristics, enabling researchers to gain insights into cell populations and their properties.
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Lab products found in correlation
Accuri c6, by BD (3 mentions)
Facsaria, by BD (3 mentions)
Facscanto 2, by BD (3 mentions)
Foxp3 transcription factor staining buffer set, by Thermo Fisher Scientific (2 mentions)
Facscelesta flow cytometer, by BD (2 mentions)
Anti tcf1 c63d9, by Cell Signaling Technology (2 mentions)
Anti id3 s30 778, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Onecomp ebeads, by Thermo Fisher Scientific (2 mentions)
Live dead aqua, by Thermo Fisher Scientific (2 mentions)
Red blood cell lysis solution, by Miltenyi Biotec (2 mentions)
Fvs780, by BD (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Accutase, by PromoCell (1 mentions)
Prism 8, by GraphPad (1 mentions)
Tumor dissociation kit, by Miltenyi Biotec (1 mentions)
F4 80 bm8, by Thermo Fisher Scientific (1 mentions)
Cd206 c068c2, by BioLegend (1 mentions)
Live dead dye, by Thermo Fisher Scientific (1 mentions)
Cellfix, by BD (1 mentions)
45 protocols using fortessa lsr
1
Flow Cytometric Analysis of Exosome Uptake
2
Multicolor Flow Cytometry Antibody Optimization
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Standard protocols were used to stain cells with directly conjugated specific, or isotype control, antibodies as described previously (Clark et al., 2016 ). Unlabeled mouse mAbs were detected with species‐specific Alexa Fluor (AF) 488 or 647F(ab’)2 secondary reagents (all from Thermo). Live cells were identified as propidium iodide‐ events. Data were collected on either an Accuri C6, Fortessa LSR, or Influx (BD Biosciences).
To determine whether different antibodies bound similar epitopes, we preincubated target cells with saturating amounts of primary antibody in 0.5% BSA/PBS for 30 min on ice. Cells were washed with 0.5%BSA/PBS before incubation with a subsaturating concentration of the test antibody. Experiments were repeated three times. Percent binding of the test antibody was determined from median fluorescence intensity (MFI) by [MFI Test Antibody‐MFI Primary isotype]/[MFI Primary Antibody‐MFI isotype control] x 100. In cross‐blocking experiments using primary samples, CD34+ HSPC, lymphocytes, and monocytes all originated from cord blood (CB) PBMC. AML samples were gated with CD45 and CD34 to exclude nonblasts.
To determine whether different antibodies bound similar epitopes, we preincubated target cells with saturating amounts of primary antibody in 0.5% BSA/PBS for 30 min on ice. Cells were washed with 0.5%BSA/PBS before incubation with a subsaturating concentration of the test antibody. Experiments were repeated three times. Percent binding of the test antibody was determined from median fluorescence intensity (MFI) by [MFI Test Antibody‐MFI Primary isotype]/[MFI Primary Antibody‐MFI isotype control] x 100. In cross‐blocking experiments using primary samples, CD34+ HSPC, lymphocytes, and monocytes all originated from cord blood (CB) PBMC. AML samples were gated with CD45 and CD34 to exclude nonblasts.
3
Apoptosis Analysis by Flow Cytometry
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Cells were fixed using neutral-buffered formalin for 10 min on ice, washed with PBS, resuspended in 0.1% Triton-X100 in PBS, and incubated for 15 min at room temperature. Permeabilized cells were washed twice with PBS, and resuspended in 100 μl of Hank's balanced salt solution (HBSS) with 0.1% bovine serum albumin and 2 μl of Alexa Fluor 647-conjugated antibody against cleaved Caspase-3. Cells were incubated for 30 min at room temperature in the dark washed twice with PBS and stained with 1 μg/ml DAPI. Cells were analyzed on the Fortessa LSR as described before (BD Bioscience) 49 (link),50 (link).
4
Single-Cell Immune Profiling in Lymphoid Tissues
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Single-cell suspensions from the spleen and lymph nodes (LNs) were generated after mashing tissue through 70 μm cell strainer, and the cells were surface-, intracellularly or intranuclearly stained following standard protocols42 (link). The fluorochrome-conjugated antibodies, including anti-CD8 (53–6.7), anti-TCRβ (H57–597), anti-CD44 (IM7), and anti-CD62L (MEL-14), anti-CD45.2 (104), anti-KLRG1 (2F1), anti-IL-7Ra (A7R34), anti-Granzyme B (GB12), anti-IFN-g (XMG1.2), anti-TNF (MP6-XT22), anti-IL-2 (JES6–5H4), anti-hNGFR (ME20.4), were from eBiosciences, ThermoFisher Scientific. Anti-Tcf1 (C63D9) was from Cell Signaling Technology, and anti-Id3 (S30–778) was from BD Biosciences. For intranuclear detection of Id3 or Tcf1, surface-stained cells were fixed and permeabilized with the Foxp3/Transcription Factor Staining Buffer Set (eBiosciences, ThermoFisher Scientific), followed by incubation with corresponding fluorochrome-conjugated antibodies. For cytokine staining, the cells were stimulated with GP33 (200 nM) in the presence of the protein transport inhibitor Brefeldin A for 5 hrs at 37°C, followed by standard intracellular staining. Cell sorting was performed on FACSAria (BD Biosciences). Data were collected on Fortessa LSR or FACSCelesta flow cytometers (BD Biosciences) and were analyzed with FlowJo software v10.7.1 (TreeStar).
5
IL-17RA Expression Profiling in Keratinocytes
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Adherent N/TERT keratinocyte cells were dissociated using Accutase (Promocell cat#41310) to obtain a single cell suspension. For the detection of cell surface expression of IL-17RA receptor on IL-17RAko, wt IL-17RA and A104E IL-17RA cells, cells were stained with Alexa Fluor® 488-conjugated anti-IL-17RA antibody (R&D Systems cat# FAB177G) or isotype control antibody Alexa Fluor488-conjugated msIgG1 (IC002F, R&D Systems) for 30 min on ice. Cells were washed once before flow cytometry analysis using a Fortessa LSR flow cytometer (BD Biosciences).
6
Apoptosis and Proliferation Assays
7
Evaluating MOMP in ALL Cells
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Evaluation of MOMP was done using the fluorescent dye tetramethylrhodamine ethyl-ester (TMRE) (Thermo Fisher Scientific). In total, 500,000 cryopreserved ALL cells were recovered for 1 h in 300 µL AIM-V medium. Upon recovery, ALL cells were treated with moxidectin (1, 2, or 3 µM) or ABT-263 (50, 100, or 250 nM) for 2 h and subsequently incubated at 37 °C for 15 min with TMRE (50 nM). After staining, the cells were collected, centrifuged, and resuspended in 1× PBS. TMRE fluorescence was quantified by flow cytometry (Fortessa LSR, BD Biosciences). Results were analyzed by FlowJo 10.2 and statistical analysis was performed using GraphPad Prism 8 with t-test analysis.
8
Multiparameter Immune Cell Analysis
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Single-cell suspensions were prepared (Tumor Dissociation Kit, 130-096-730, Miltenyi Biotec, Paris, France) and incubated with fluorophore-conjugated anti-mouse antibodies (CD45 [30-F11], CD3 [17-A2], CD4 [RM4-5], CD8 [53-6.7], CD11b [M1/70], Ly-6g [1A8]; all from BD Biosciences, Le Pont de Claix, France), CD206 (C068C2) (from BioLegend EU Amsterdam, NL), and F4/80 (BM8; from ThermoFisher Scientific). Data acquisition was performed on the Fortessa LSR (BD Biosciences) and BD FacsDiva software was used for analysis, as published previously.47
9
Multi-color Flow Cytometry Immunophenotyping
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Four lasers and 9 different PMT channels were utilised for the 9-colour staining panel; cells were stained with antibodies as detailed in Table 1 . Briefly, cells were washed and stained with Live/Dead dye (eBioscience) in PBS. Cells were blocked with anti-CD32 to prevent Fc-mediated non-specific binding as per manufacturer’s instructions. Cells were then stained with antibodies in buffer containing PBS, 1% Bovine Serum Albumin (BSA) and 0.1% Sodium Azide at 4°C for 30 mins followed by further washing and cell fixation (BD Cell Fix). For monocyte surface antigen characterisation, cells were stained with the baseline antibodies detailed in Table 2 . Cells were then stained with all antibodies listed in Panel 1 and each sample individually with a Mab listed in Panel 2. Flow cytometric compensation was performed using fluorescent compensation beads (OneComp eBeads, eBioscience USA) and cells were analysed using a multi-parameter flow cytometer (Fortessa LSR BD Biosciences, USA). Cell sorting was performed using a Fortessa Aria (BD Biosciences, USA). For identification of positive and negative populations, the fluorescence minus one (“FMO”) principle was utilised to account for background antibody fluorescence.
10
Detection of SARS-CoV-2 Spike-Specific Memory B Cells
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Detection of antigen-specific memory B cells was performed with the SARS-CoV-2 Spike B cell analysis kit (Miltenyi Biotec #130-128-022) at the Ospedale Pediatrico Bambino Gesù (Rome). Briefly, the kit allows the formation of tetramers made of a recombinant SARS-CoV-2 Spike-Protein (expressed in HEK)-Biotin with either Streptavidin-PE or Streptavidin-PE-Vio770. ∼5x106 previously frozen PBMC samples were prepared and stained with antibody staining mix containing the Spike tetramers, the 7-AAD and the fluorochrome-conjugated antibodies (CD19 APC-Vio770 clone LT19, CD27 Vio Bright FITC clone M-T271, CD24 BV711 clone ML5, IgA VioGreen clone IS11-8E10, IgM APC clone Pj2-22H3, IgG VioBlue clone IS11-3B2.2.3-all from Miltenyi; CD24 BV711 clone ML5 from BD). Memory B cells were identified as CD19+CD24+CD27+ and Spike-specific memory B cells were double-positive for PE and PE-Vio770 (Spike++). Samples were acquired on Fortessa LSR (BD) (BD) and analyzed using FlowJo (version 10.7.1, BD). Limit of detection (LOD) and limit of quantification (LOQ) were calculated as previously reported.58 (link),59 (link),60 (link),61 (link) Briefly, LOD was calculated as 20x100/total no. of events and LOQ was computed as 30x100/total no. of events. Gating strategy is illustrated in Figure S12 .
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