Cellular immunotyping through flow cytometry (8 color Gallios flow cytometry, Beckman Coulter, France) was made using the peripheral blood obtained by venipuncture, with the use of the K2-EDTA anticoagulant and the lysing solution VersaLyse (BeckmanCoulter, France). According to the recommendations of the manufacturer (Beckman Coulter, France), we used the red blood cell lysis without washing. To each volume of the conjugate, we added 100 μL of blood, mixed for 3 s, and incubated it in a dark chamber for 15 min. at room temperature. Then we added 1 mL of the lysis buffer VersaLyse TM (Beckman Coulter, France) and incubated it for 10 min under the same conditions as the previous step. Finally, we immediately proceeded to the acquisition of the sample by the cytometer. The acquisition of the data was carried out through the Kaluza Acquisition v1.0 software by which we obtained a minimum of 50,000 total events. For the analysis and the report of the results we used Kaluza Analysis v1.5a. The absolute cell counts of the lymphocyte populations were performed through a dual-platform. We designed a manual and sequential window selection strategy with bi-parametric graphs (Fig. S1 ). For the reference values of the analyses of cellular sub-populations we used previous studies in the Cuban population (Kokuina et al, 2019 (link)).
Versalyse
Manufactured by Beckman Coulter
Sourced in United States, France, Germany, Japan
VersaLyse is a lyse reagent designed for the preparation of cell samples for analysis by flow cytometry. It is used to lyse red blood cells, while preserving the integrity of the target cells, to enable the analysis of cell populations within a whole blood sample.
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Lab products found in correlation
Navios, by Beckman Coulter (4 mentions)
Cytoflex flow cytometer, by Beckman Coulter (3 mentions)
Navios flow cytometer, by Beckman Coulter (3 mentions)
Trypsin edta 0.25, by Thermo Fisher Scientific (2 mentions)
3 isobutylmethylxanthine, by Merck Group (2 mentions)
Penicillin streptomycin pen strep broad spectrum antibiotic cocktail, by Thermo Fisher Scientific (2 mentions)
Collagenase type 1, by Thermo Fisher Scientific (2 mentions)
Dexamethasone (dex), by Merck Group (2 mentions)
Indomethacin, by Merck Group (2 mentions)
Navios cytometer, by Beckman Coulter (2 mentions)
Nigericin, by InvivoGen (2 mentions)
Cd14 apc, by Beckman Coulter (2 mentions)
Cd45 pb, by Beckman Coulter (2 mentions)
Anti asc pe, by BioLegend (2 mentions)
Iotest fixative solution, by Beckman Coulter (2 mentions)
Iotest 3 fixative solution, by Beckman Coulter (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Fc500 flow cytometer, by Beckman Coulter (2 mentions)
Attune nxt, by Thermo Fisher Scientific (2 mentions)
Kaluza analysis 2, by Beckman Coulter (1 mentions)
50 protocols using versalyse
1
Cellular Immunotyping by Flow Cytometry
2
Flow Cytometric Profiling of Cryopreserved Patient Cells
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Flow cytometry surface and intracellular stainings of cryopreserved patient material were conducted as described before (11 (link)). To assess phosphorylation of signal transducers, PBMCs were either short term stimulated by 10 µg/mL soluble αCD3+/αCD28+ for 15 min or remained unstimulated at 37 °C/5% CO2 in a density of 5 × 105 cells/well. For CSF staining, CSF specimen was processed within 1 h from lumbar puncture by centrifugation at 4 °C for 15 min at 290 g. The samples were treated with VersaLyseTM (Beckman Coulter) for 10 min at room temperature (RT). A full list of used antibodies, fluorochromes, working concentrations, and company names is provided in Dataset S15 . All investigated immune cell populations with corresponding gating strategies per population and experiment are listed in Dataset S16 . Immune cells were acquired by Navios or Gallios (Beckman Coulter) flow cytometer and analyzed by Kaluza Analysis 2.1 (Beckman Coulter).
3
Optimizing Mesenchymal Stem Cell Isolation
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Collagenase type I, penicillin/streptomycin (Pen/Strep) broad-spectrum antibiotic cocktail, trypsin-EDTA (0.25%), fetal bovine serum (FBS), human insulin and Dulbecco’s Modified Eagle’s Medium (DMEM) were purchased from Gibco/Invitrogen (Carlsbad, CA, USA). VersaLyseTM was purchased from Beckman Coulter (Miami, FL, USA). Dexamethasone, 3-isobutyl-methylxanthine, Nile Red (NR) and indomethacin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vybrant® DyeCycleTM Violet was purchased from Thermo Fisher Scientific/Life Technologies (Eugene, OR, USA). The following mouse anti-human monoclonal antibodies were purchased from Biolegend (San Diego, CA, USA): CD14-APC Cy7 (Clone M5E2), CD31-PE Cy7 (Clone WM-59), CD36-APC (Clone 5-271), CD73-FITC (Clone AD2), CD44-APC Cy7 (Clone IM7) and CD105-PE (Clone 42A3). Mouse anti-human CD45-Krome Orange (Clone J.33), CD90-PE-Cy5 (Clone Thy-1), CD34-PE Cy7 (Clone 581), and the viability dye, 7-aminoactinomycin D (7-AAD) were purchased from Immunotech/Beckman Coulter (Marseille, France).
4
Flow Cytometry Analysis of CSF and Peripheral Blood
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Flow cytometric measurements were done in one study centre (Muenster). For this purpose, PB and CSF were analysed within one hour after sampling. The CSF was centrifuged and treated with VersaLyseTM (Beckman Coulter, Krefeld, Germany) in parallel to PB, according to the manufacturer’s instructions. Cells were incubated with fluorochrome-conjugated monoclonal antibodies [CD14-FITC (clone RM052), CD138-PE (clone B-A38), HLA-DR-ECD (clone Immu-357), CD3-PC5.5 (clone UCHT1), CD56-PC7 (clone N901), CD4-APC (clone 13B8.2), CD19-APC A700 (clone J3-119), CD16-APC A750 (clone 3G8), CD8-PacificBlue (clone B9.11), CD45-KromeOrange (clone J.33); all Beckman Coulter, dilution 1:200] for 30 min, washed and analysed by flow cytometry on a NaviosTM (Beckman Coulter) flow cytometer.32 (link) Data were analysed with KaluzaTM 2.1 software (Beckman Coulter; for gating strategy, see Supplementary Fig. 1 ). Routine CSF parameters were investigated in addition to flow cytometry in both study centres (Magdeburg and Muenster). Cells were counted using a Fuchs-Rosenthal chamber. The CSF/serum IgG, IgA, IgM, and albumin ratio and the blood/CSF-barrier integrity were determined by nephelometry (BN ProSpecTM, Siemens Healthcare). IgG oligoclonal band (OCB) patterns were analysed by isoelectric focussing in gel-electrophoresis and subsequent silver staining (Processor PlusTM, GE Healthcare).
5
Monocyte Subset Analysis by Flow Cytometry
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Whole-blood samples (200μL) were labeled with anti-CD14-PC7 (clone RMO52); CD16-PB (clone 3G8); CD2-FITC (clone 39C1.5); CD56-PC5.5 (clone N901); CD24-PE (clone ALB9); CD45-KO (clone J33) and HLA-DR-APC (clone Immu-357) antibodies, all purchased from Beckman-Coulter. Red blood cells were lysed with 1 mL VersalyseTM (Beckman Coulter) before sample analysis with a Navios Cytometer (Beckman Coulter) as described (Tarfi et al., 2019 ). Monocytes were selected as CD45High/SSCInt cells among living cells and singlets before excluding T cells as CD2+/SSCLow, NK cells as CD56+/SSCLow/Int, B cells as CD24+/SSCLow, immature and mature granulocytes as CD24+/SSCInt/High, CD16Bright residual granulocytes, and remaining CD14−CD16− cells corresponding mainly to basophils and NK cells not previously excluded. Monocyte subsets were detected on a CD45/SCC dot plot, using a CD14/CD16 scattergram that separates CD14HighCD16Low (classical), CD14High/CD16High (intermediate) and CD14LowCD16High (non-classical) subsets. Finally, the proportion of monocytes HLA-DRLow was evaluated on a HLA-DR/CD14 scattergram.
6
Flow Cytometry Analysis of Immune Cells
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The following antibodies were used in flow cytometry experiments of whole blood: CD3-FITC (clone UCHT1), CD56-PE (clone HCD56), CD16-APC (clone 3G8), all from Biolegend, and CD59-APC (clone MEM-43) from ImmunoTools. Unconjugated CD16-specific monoclonal antibodies 3G8 and B73 (purified from hybridoma supernatants by affinity chromatography using Protein G-Sepharose (GE Healthcare), hybridomas were a gift of Prof JL Strominger), followed by goat anti-mouse secondary antibodies coupled to FITC (Dakocytomation) were used to stain the transfected HEK-293T cells (the two mAbs gave similar results). Unlabelled IgG1 and IgG2a isotype control mAbs were purchased from Sigma Aldrich, while fluorescently labelled isotype control antibodies were purchased from Biolegend.
Most experiments were carried out by staining 100 µl of whole blood with directly labelled mAb. Blood samples were washed in PBS buffer containing 0.5% (w/v) bovine serum albumin, 1% (v/v) foetal bovine serum, 0.1% sodium azide, and stained with labelled mAbs specific for the indicated receptors for 40 min at 4°C. Red blood cells were lysed with 1 ml VersalyseTM (Beckman Coulter) for 10 min before analysis in FACSCalibur (BD Biosciences) cytometer. Data were analysed with FlowJo and Kaluza Flow Cytometry Analysis programs.
Most experiments were carried out by staining 100 µl of whole blood with directly labelled mAb. Blood samples were washed in PBS buffer containing 0.5% (w/v) bovine serum albumin, 1% (v/v) foetal bovine serum, 0.1% sodium azide, and stained with labelled mAbs specific for the indicated receptors for 40 min at 4°C. Red blood cells were lysed with 1 ml VersalyseTM (Beckman Coulter) for 10 min before analysis in FACSCalibur (BD Biosciences) cytometer. Data were analysed with FlowJo and Kaluza Flow Cytometry Analysis programs.
7
Murine Immune Cell Phenotyping
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Peripheral blood cells or splenocytes harvested from mice and processed to produce single-cell suspensions were stained with antibodies to CD3 (145-2C11), CD4 (RM4-5), CD25 (PC61), CD8 (53-6.7), B220 (RA3-6B2), CD44 (IM7), and CD62L (MEL14) (BD Biosciences, San Jose, CA, USA). Class II MHC tetramers included MOG38–49/I-Ab class II MHC (GWYRSPFSRVVH) and h.CLIP87–101 (PVSKMRMATPLLMQA), and were provided by the NIH Tetramer Core (Emory University, Atlanta, GA, USA). Red blood cell lysis was performed with VersaLyse (Beckman Coulter, Brea, CA, USA). Intracellular staining for FOXP3 was performed using the FOXP3 staining kit (eBioscience, San Diego, CA, USA). Samples were analyzed on an LSR-II flow cytometer (BD Biosciences) and post-analyzed using FCS Express 4 (Denovo Software, Los Angeles, CA, USA).
8
Multiparametric Flow Cytometry Analysis
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CD45RA-FITC and CD45RO-PE antibodies were obtained from BD Biosciences (San Diego, CA). CD38-APC and CD4-PerCP antibodies were obtained from BioLegend (San Diego, CA). Fresh peripheral blood mononuclear cells were stained using antibodies cocktail in 20 min at room temperature, and then, red cells were lysed in 15 min with Versalyse (Beckman Coulter, Brea, CA). Flow cytometry and data analysis were, respectively, performed on a BD CANTO II (BD Biosciences, San Diego, CA).
9
Immunophenotyping of Immune Cells
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The following antibodies were used: CD45-PB, CD14-APC (both from Beckman Coulter, Brea, CA, USA, clones J33 and RMO52, respectively), CD15-AF700 and anti-ASC-PE (both from Biolegend, San Diego, CA, USA, clones W6D3 and HASC-71, respectively). Following reagents were used: phosphate-buffered saline (PBS) (Eurobio Scientific, Luxembourg, Luxembourg), Ficoll (Dutscher, Bruxelles, Belgium), RPMI HEPES (Eurobio, Les Ulis, France), penicilline/streptomycine (Clinisciences, Nanterre, France), fungizone (Cheplapharm Arzneimittel, Mesekenhagen, Germany), glutamine (Ozyme, Saint-Cyr-l’École, France), versalyse (Beckman Coulter), lipopolysaccharide (LPS) (Sigma-Aldrich, single mix of references L3137, L2637 and L3012, Saint-Louis, MO, USA), nigericin (InvivoGen, San Diego, CA, USA), water for injection (Aguettant, Lyon, France), Cytofix/Cytoperm, Fixation/Permeabilization Kit, stain buffer (both from BD Bioscience, Franklin Lakes, NJ, USA) and human AB serum (SAB) (“Etablissement Français du Sang”, Lyon, France).
10
Comprehensive Immune Cell Profiling
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All analysis was performed using DuraClone IM (Immune Monitoring) basic antibody panel (CD45, CD3, CD4, CD8, CD19, CD14, CD16, CD56) (9 (link)). For peripheral blood analysis, 100 μl of blood was added to the basic panel tube and cells were processed according to the manufacturer’s instructions. In brief, blood was incubated with the antibodies for 15 min in the dark, followed by red blood cell lysis using VersaLyse (Beckman Coulter) for 15 min in the dark. Cells were then washed twice in PBS prior to data acquisition. For tumor cells and tumor-infiltrating immune cells, 1 × 106 cells were added to the basic panel tube and cell staining was performed as described above for peripheral blood. Additionally, propidium iodide (PI, Sigma-Aldrich, St. Louis, MO) was added as a live-dead marker to tumor single cell suspensions. All processed samples were then analyzed on a 13-color CytoFlex flow cytometer (Beckman Coulter). Data was analyzed using Kaluza Software (Beckman Coulter) as previously described (9 (link)).
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