For the flow cytometry analyses on non‐cultured cells, we let the cells recover after enzymatic cell isolation for 24 hours before continuing with the FACS procedure because the enzymes used for cell isolation affects the presence of the cell surface markers (data not shown). For FACS analyses on cultured cells, the cells directly after detaching with accutase at passage 3 or 4 were used. The cells were fixed in BD Cytofix/Cytoperm (BD biosciences, USA) after staining with the Zombie violet fixable viability kit (BioLegend, USA) to identify dead cells and select for viable cells during analyses. Fixed cells were stained with fluorochrome‐conjugated antibodies to detect the presence of the various SLC markers. We titrated all antibodies first to determine the correct antibody concentration for FACS analyses. Details of the used antibodies for FACS analyses are described in Table S2 . FACS analyses were performed on the BD FACSCanto II Flowcytometer or the BD LSRFortessa cell analyzer flow cytometer. FlowJo 10.2 software was used to analyze the data and GraphPad Prism 5 program to make the graphs. All experiments were reproduced on at least three donors. Matched isotype controls were used to determine background fluorescence. The flow cytometry data were expressed as the mean percentage of positive cells ± SEM.
Lsrfortessa cell analyzer flow cytometer
Manufactured by BD
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The BD LSRFortessa cell analyzer is a flow cytometer that enables the analysis and sorting of cells. It provides high-performance data acquisition and analysis capabilities for a wide range of applications.
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Lab products found in correlation
Facscanto 2 flow cytometer, by BD (3 mentions)
Cytofix cytoperm, by BD (3 mentions)
Facsdiva v6, by BD (3 mentions)
Zombie violet fixable viability kit, by BioLegend (2 mentions)
Brefeldin a, by BioLegend (2 mentions)
Fixation buffer, by BioLegend (2 mentions)
Monensin solution, by BioLegend (2 mentions)
Permeabilizing buffer, by BioLegend (2 mentions)
Legendplex software, by BioLegend (1 mentions)
Legendplex mouse inflammation panel kit, by BioLegend (1 mentions)
Facsdiva software, by BD (1 mentions)
Live dead fixable aqua dead cell stain, by Thermo Fisher Scientific (1 mentions)
Cellrox green reagent, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Cytometric bead array cba human th1 th2 cytokine kit, by BD (1 mentions)
Sh800z cell sorter, by Sony (1 mentions)
7 aad, by BD (1 mentions)
Cd105, by BD (1 mentions)
Pdgfrα, by BD (1 mentions)
Facsdiva 8, by BD (1 mentions)
23 protocols using lsrfortessa cell analyzer flow cytometer
1
Flow Cytometry Analysis of Cell Surface Markers
2
FACS Analysis of Cultured Cells
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For FACS analysis, the cells were cultured in vitro and amplified. TSCs or ADSCs were harvested and suspended in PBS (1 × 106 cells/mL), and then all these cells were incubated in BD Cytofix/Cytoperm (BD Biosciences, USA). We stained the cells with fluorochrome-conjugated antibodies in the dark for 30 min at 4 °C. The samples were centrifuged at 1100 rpm at 4 °C for 4 min. Then, the pellet was washed with PBS and finally analyzed by BD FACSCanto II Flow cytometer (BD Biosciences, USA) or the BD LSRFortessa cell analyzer flow cytometer (BD Biosciences, USA).
3
Cytokine Profiling of Immune Cell Responses
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RAW 264.7 and P388.D1 cells were seeded on a 96-well plate (3 × 104 cells per well in 100 µL DMEM, 5% (v/v) FBS). After 16 h, the medium was replaced with DMEM supplemented with 2% (v/v) FBS and 1) 100 ng/mL LPS, 2) 10 ng/mL IFN-γ, 3) 1 μM BacSp222, 4) 1 μM suc-K20-BacSp222, 5) 1 μM suc-K11/K20-BacSp222, 6) 1 μM -fM-BacSp222, 7) 1 μM nisin A, and 8) 10 ng/mL INF-γ combined with 100 ng/mL LPS or 1 μM bacteriocins. After 24-h stimulation, the cultured media were collected, and cytokine concentrations (IL-1α, IL-1β, IL-6, IL-10, IL-12p70, IL-17α, IL-23, IL-27, MCP-1, IFN-β, IFN-γ, TNFα, and GM-CSF) were determined using a LEGENDplex Mouse Inflammation Panel kit (Biolegend, San Diego, CA, USA) and a BD LSRFortessa Cell Analyzer flow cytometer (BD Bioscience, Franklins Lake, NJ, USA). The results were analyzed using LEGENDplex software (Biolegend, San Diego, CA, USA).
4
Flow Cytometry-based Orthogonal Drug Assay
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Cell cultures were
titrated with various concentrations of the appropriate orthogonal
drugs. 1.5 μL of different concentrations of 100× concentrated
drug solution (TMP in Dimethyl sulfoxide (DMSO) and/or Shld-1 in absolute
Ethanol) was added to the wells to achieve the final concentration.
For cultures without any drug, corresponding volumes of solvents (DMSO
and/or Ethanol) were added. The plates were incubated for 72 h before
harvesting for measuring the fluorescence by flow cytometry: fluorescence
measurements were performed on a BD LSRFortessa cell analyzer flow
cytometer. The eGFP fluorescence was measured using a 488 nm excitation
laser and a 515–545 nm emission filter, while mCherry fluorescence
used 561 nm excitation and 600–620 nm emission. A minimum of
10 000 cells was measured from each sample. From these single-cell
fluorescence intensities, we further computed the mean fluorescence
intensity per cell representing the population average for both mCherry
and eGFP separately using the FlowJo software (Treestar, Inc., San
Carlos, CA). The mean eGFP fluorescence values were normalized to
mCherry fluorescence intensities after subtracting for autofluorescence
derived from mock-transfected cells. The resulting ratiometric scores
were further converted to %, based on the ratiometric score of the
control eGFP, without DDs or drug, but with the respective solvents
of the drugs.
titrated with various concentrations of the appropriate orthogonal
drugs. 1.5 μL of different concentrations of 100× concentrated
drug solution (TMP in Dimethyl sulfoxide (DMSO) and/or Shld-1 in absolute
Ethanol) was added to the wells to achieve the final concentration.
For cultures without any drug, corresponding volumes of solvents (DMSO
and/or Ethanol) were added. The plates were incubated for 72 h before
harvesting for measuring the fluorescence by flow cytometry: fluorescence
measurements were performed on a BD LSRFortessa cell analyzer flow
cytometer. The eGFP fluorescence was measured using a 488 nm excitation
laser and a 515–545 nm emission filter, while mCherry fluorescence
used 561 nm excitation and 600–620 nm emission. A minimum of
10 000 cells was measured from each sample. From these single-cell
fluorescence intensities, we further computed the mean fluorescence
intensity per cell representing the population average for both mCherry
and eGFP separately using the FlowJo software (Treestar, Inc., San
Carlos, CA). The mean eGFP fluorescence values were normalized to
mCherry fluorescence intensities after subtracting for autofluorescence
derived from mock-transfected cells. The resulting ratiometric scores
were further converted to %, based on the ratiometric score of the
control eGFP, without DDs or drug, but with the respective solvents
of the drugs.
5
Immunophenotypic Characterization of PL-MSCs
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At passages 4–5, detach the PL-MSCs with TrypLE and calculate the cell number as per Step 19.
Wash the harvested cells 1–2 times with ice-cold PBS by centrifuging at 500 × g for 2 min.
Discard the supernatant and suspend the cells in PBS at 2 × 106 cells/ml.
Aliquot 1 × 105 cells or 50 μL of cell suspension in each of the pre-labeled 5 mL FACS tubes.
Add the respective antibodies to the tubes and incubate in ice for 1 h. Mouse isotype antibodies serve as controls.
Acquire at least 10,000 events on BD LSRFortessa Cell Analyzer flow cytometer and analyze the results using BD FACSDiva software.
Representative data from a PL-MSC culture at passage 4 is presented in Immunophenotypic characterization of isolated PL-MSCs The antibodies used were (A) human CD73, (B) human CD90 (C) human CD105 (D) human CD166 (E) human CD34 (F) human HLA-DR and their respective isotype controls. Open histogram indicates background signal, while, shaded histogram represents positive reactivity with the indicated antibodies. Table shows percentage of cells expressing the respective surface markers (n = 3) in mean ± SEM.
6
SARS-CoV-2-Specific B Cell Detection
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For the detection of SARS-CoV-2-specific B cells, the PBMC were stained with LIVE/DEAD Fixable Aqua Dead Cell Stain (Life Technologies), anti-CD14-BV510 (clone M5E2), anti-CD3-BV510 (clone UCHT1), anti-CD56-B510 (clone NMCAM16.2), anti-CD38-BV605 (clone HB7), anti-IgM-BV650 (clone G20-127), anti-IgG-BV421 (clone G18-145), anti-CD19-APC (clone SJ25C1), anti-IgD-AF700 (clone IA6-2), anti-CD20-PeCy7 (clone L27), anti-CD27-Pe (clone L128) (all of them from BD Bioscience) and anti-SARS-Cov-2 S protein-AF488 (clone P05DHu) (Abcam). In addition, the SARS-CoV-2 protein conjugated to AF488 was used to detect SARS-CoV-2 specific B cells. Briefly, the SARS-CoV-2 S1/S2-protein (also referred to as S-protein) (Sino Biological Inc.) was coupled with Alexa Flour 488 fluorochrome using the Lightning-Link® Rapid Conjugation System (Abcam) according to the manufacturer’s protocol. B cells were gated according to CD3, CD14, CD56, and CD19 expression. B cell subsets [(naïve, memory, and antibody-secreting cells (ASC)] were gated based on IgD, CD27, and CD38 expression. The samples were acquired on a BD LSR Fortessa™ Cell Analyzer flow cytometer using FACS Diva software (BD Biosciences) and analyzed using FlowJo 10.7.1 software (Treestar, Ashland, OR). Supplementary Figure 2
7
Quantifying Cellular Reactive Oxygen Levels
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To measure reactive oxygen species (ROS) levels, we used the CellROX Green Reagent (Thermo Scientific) according to the manufacturer’s protocol. Menadione treatment (100 μM for 1 h at 37 °C) was used as a positive control. After labeling with CellROX Green Reagent, samples were collected by trypsinization, washed 1× in PBS (500g, 5 min), fixed in 4% paraformaldehyde (RT, 15 min) and stored at 4 °C. Samples were measured the subsequent day. ROS levels were quantified by recording green fluorescence (FITC/GFP detector) of at least 20,000 single cells per sample, on a BD LSR Fortessa Cell Analyzer flow cytometer (BD Biosciences), and the data were analyzed using FlowJo (V10.1.1).
8
Cytofluorometric Analysis of Ploidy
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Samples for cytofluorometric analysis of ploidy were taken from embryos and their foetal membranes, from the bone marrow of newborn mice and tissues of adult animals after homogenisation and trypsinisation. Bone marrow was flushed out from femora using fine syringes. All cell suspensions were rinsed with culture medium, then twice with PBS and then with ice-cold 70% ethanol and were centrifuged 5 min at 1000 r.p.m. between each rinse. Finally, the pellets were resuspended in ice-cold ethanol for fixation and stored at 4 °C. Before cytofluorometric analysis, the samples were rinsed with PBS and stained with Hoechst 33342 (5 μg/mL, Molecular Probes, Thermofisher, Grand Island, NY, USA).
A BD LSR Fortessa Cell Analyzer flow cytometer (BD Biosciences, San Jose, CA, USA) was used. Chimaeric embryos were analysed for the presence of fluorescent introduced cells and their ploidy.
A BD LSR Fortessa Cell Analyzer flow cytometer (BD Biosciences, San Jose, CA, USA) was used. Chimaeric embryos were analysed for the presence of fluorescent introduced cells and their ploidy.
9
Th1/Th2 Cytokine Profiling in Raw 264.7 Cells
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BD™ Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit (BD-Biosciences) was used to measure Th1/Th2 inflammatory cytokines. Raw 264.7 cells were seeded at 2 × 10 5 cells/mL into 48-well plates. After 24 hours, cells were either left untreated or treated with samples and stimulated with LPS 0.1 μg/mL Indomethacin (14 μM) was used as a positive control. After 24 hours of incubation, cell culture supernatant was collected, and the level of IL-2, IL-4, IL-6, IL-10, IFN-γ and TNF-α protein was quantified by multiplex cytokine array analysis. Data were acquired on a BD LSR Fortessa™ cell analyzer flow cytometer.
10
Comprehensive Flow Cytometry Analysis of MSCs
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For flow cytometry analysis we used the BD FACSCanto II Flowcytometer (BD Biosciences, USA) or the BD LSRFortessa cell analyzer flow cytometer (BD Biosciences, USA) in combination with the FlowJo 10.2 software (FlowJo, LLC, USA) to analyze the data. For FACS analysis, cells were fixed in BD Cytofix/Cytoperm (BD Biosciences, USA). We stained the cells with fluorochrome-conjugated antibodies for the detection of the cell surface markers PDGFRα (BD Biosciences, USA), CD90 (eBioscience, Thermo Fisher Scientific, USA), CD73 (BD Biosciences, USA) and CD105 (BD Biosciences, USA). According to the minimal criteria for MSCs, cells need to be positive for CD90, CD73 and CD105 to describe them as MSCs (Dominici et al., 2006 (link)). To exclude non-viable cells, we used the Zombie violet fixable viability kit (Biolegend, USA) to stain dead cells before the cells were fixed.
Non-fixed ICF cells were sorted at Passage 3 using the SH800Z Cell Sorter (Sony Biotechnology, USA) for the HLA-A,B,C+/CD34−/PDGFRα+ subpopulation to exclude germ cells and endothelial cells from the sorted population. Non-viable cells were excluded by the use of 7-AAD (BD Biosciences, USA). For both analysis and sorting, we determined background fluorescence levels by using matched conjugated IgG isotype controls. The details of the antibodies that were used can be found inSupplementary Table SI .
Non-fixed ICF cells were sorted at Passage 3 using the SH800Z Cell Sorter (Sony Biotechnology, USA) for the HLA-A,B,C+/CD34−/PDGFRα+ subpopulation to exclude germ cells and endothelial cells from the sorted population. Non-viable cells were excluded by the use of 7-AAD (BD Biosciences, USA). For both analysis and sorting, we determined background fluorescence levels by using matched conjugated IgG isotype controls. The details of the antibodies that were used can be found in
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