Fluorochrome-conjugated anti-human CD2 (RPA-2.10), anti-CD4 (RM4-5), anti-CD8α (53-6.7), anti-CD11c (N418), anti-CD45R (RA3-6B2), anti-CD62L (MEL-14), anti-F4/80 (BM8), anti-Foxp3 (FJK-16S), anti-IFNγ (XMG1.2), anti-IL-10 (JES5-16E3), and anti-IL-17A (eBio17B7) antibodies were purchased from BioLegend and eBioscience. Prior to cytokine staining, cells were stimulated with phorbol 12-myristate 13-acetate (PMA, 10 ng/mL) and Ionomycin (0.5 μg/mL) for 4 h, with Brefeldin A (10 μg/mL) added for the final two h (all Sigma-Aldrich). Surface staining was performed for 15 min on ice in PBS (Gibco) containing 0.2 % bovine serum albumin (BSA, Sigma-Aldrich). Intracellular Foxp3 staining was performed using Foxp3 staining buffer set (eBioscience) according to the manufacturer's instructions. Absolute cell numbers were determined using Accurri C6 Cytometer (Becton Dickinson). Dead cell exclusion was carried out using LIVE/DEAD Fixable Blue Dead Cell Stain (Thermo Fisher Scientific). Cells were washed, resuspended in phosphate buffered saline/bovine serum albumin (PBS/BSA), and measured at LSR-II SORP with Diva software 6.1 (BD Biosciences). Data were analyzed using FlowJo software (Tree Star).
Diva software 6
Manufactured by BD
Sourced in United States
Diva software 6.0 is a laboratory data management software developed by BD. It provides a platform for the storage, analysis, and reporting of data generated from various laboratory instruments and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto flow cytometer, by BD (2 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (2 mentions)
Accurri c6 cytometer, by BD (1 mentions)
Live dead fixable blue dead cell stain, by Thermo Fisher Scientific (1 mentions)
Brefeldin a, by Merck Group (1 mentions)
Foxp3 staining buffer set, by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Facs lsr 2, by BD (1 mentions)
Streptavidin qdot605, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Carl Roth (1 mentions)
Cd105 pe, by Thermo Fisher Scientific (1 mentions)
Cd14 fitc, by BD (1 mentions)
Cd34 phycoerythrin, by BD (1 mentions)
Cd79a apc, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Cd73 pe, by BD (1 mentions)
A2754, by Merck Group (1 mentions)
D7008, by Merck Group (1 mentions)
Facscanto 2 flow cytometer, by BD (1 mentions)
Cd45ra pe cy7, by BioLegend (1 mentions)
8 protocols using diva software 6
1
Multiparametric Flow Cytometry Analysis
2
Multicolor Flow Cytometry of Immune Cells
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Cells were stained with anti-mouse CD19-BV650 (6D5), B220-BV510 (RA3-6B2), CD93-PerCPC5.5 (AA4.1), CD138-BV421 (281-2), c-kit-AlexaFlour700 (ACK4), IgK-PE (RMK-45), IgG1-bio (RMG1-1), IgG2a-bio (RMG2a-62), IgG2b-bio(RMG2b-1) (BioLegend, San Diego CA, USA) and CD25-PeCy7 (eBio3C7), IgM-APC or -PeCy7 (II/41), TACI-APC (ebio8F10-3) (eBioscience, San Diego CA, USA) in the presence of anti-FCgRII (2.4G2, inhouse). Streptavidin-Qdot605 (Molecular Probes) was used to visualize biotin-conjugated primary antibodies. Lymphocates were analyzed using an LSRII FACS (BD Biosciences) in the presence of DAPI (Carl Roth GmbH). Aggregates and doublets were gated out [4] . Acquisition was performed using the DiVa software 6.1 (BD Biosciences). Analysis was performed using the FlowJo software (Tree Star, Ashland OR, USA).
3
Immunophenotypic Characterization of MSCs
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At P3, MSCs from both sources (1 × 107 cells) were digested with trypsin and washed twice with phosphate-buffered saline. The cell concentration was adjusted to 2 × 106 cells/mL, and cells were stained with the following fluorescent antibody conjugates: CD45-fluorescein isothiocyanate (FITC), CD34-phycoerythrin (PE), CD73-PE, CD14-FITC, CD79a-APC, the human major histocompatibility complex (MHC) class II molecule HLA-DR-(PE), CD90-allophycocyanin (APC) (BD Biosciences, MD, USA), and CD105-PE (eBioscience, CA, USA). We also tested for the co-inhibitory molecule B7-H1(FITC) and the positive co-stimulatory factors CD80-PE, CD83-APC, and CD86-FITC. Surface staining was detected using flow cytometry (Diva software 6.0, FACScantoII, BD Biosciences).
4
Platelet Aggregation and ROS Measurement
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PRP samples (adjusted to 2 × 107 platelets/mL) were stimulated with the adenosine 5′-diphosphate (ADP, 16.7 µM, Sigma Aldrich A2754, Burlington, MA, USA) or phorbol 12-myristate 13-acetate (PMA, 100 µM, Sigma Aldrich 79346, Burlington, MA, USA). For ADP stimulation, the recording time was 10 min, and for PMA, the recording time was 15 min (AgreGO, Sao Paulo, Brazil). The degree of aggregation was expressed as a percentage of the maximum light transmission obtained with PRP [23 (link)].
DHE probe (500 µM, Sigma Aldrich D7008, Burlington, MA, USA) was added to 200 µL of the platelet suspension, protected from light, and placed at 37 °C for 30 min. After washout, the fluorescence was evaluated with a flow cytometer FACS CANTO II equipped with a 15-mW argon laser, λ = 488 nm (BD, Santa Monica, CA, USA). A total of 10,000 events were acquired in the FITC channel (564–606 nm) [21 (link),22 (link)], followed by analysis using the DIVA software 6.0 (BD, Santa Monica, CA, USA).
DHE probe (500 µM, Sigma Aldrich D7008, Burlington, MA, USA) was added to 200 µL of the platelet suspension, protected from light, and placed at 37 °C for 30 min. After washout, the fluorescence was evaluated with a flow cytometer FACS CANTO II equipped with a 15-mW argon laser, λ = 488 nm (BD, Santa Monica, CA, USA). A total of 10,000 events were acquired in the FITC channel (564–606 nm) [21 (link),22 (link)], followed by analysis using the DIVA software 6.0 (BD, Santa Monica, CA, USA).
5
Multiparametric Flow Cytometry Panel
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0.8–1 × 106 MNCs were stained with antibodies for 30 min at 4° C in PBS with 0.1% bovine serum albumin (BSA, Applichem, Darmstadt, Germany), followed by live/dead staining with fixable viability dye eFluor-780 (eBioscience, Frankfurt, Germany) in PBS for additional 30 min at 4° C. Cells were resuspended in 200 µL PBS + 0.1% BSA for flow cytometry. The following antibodies were used: CD3-FITC (clone BW264/56, Miltenyi Biotec, Bergisch–Gladbach, Germany); CD8-V500 (clone RPA-T8), CD45RO-FITC (clone UCHL1), CD62L-V450 (clone DREG-56), (both BD Bioscience, Heidelberg, Germany); CD45RA-PE-Cy7 (clone HI10 Biolegend, Koblenz, Germany); CCR7-PE (clone 150503, BioTechne, Wiesbaden, Germany); PD-1-APC (clone eBioJ105),TIM-3-PerCP-eFluor710 (clone F38-2E2, both eBioscience). Cells were analyzed on a FACSCanto II using the Diva software 6.0 (BD Bioscience).
6
Apoptosis Analysis by Flow Cytometry
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Apoptosis analysis was based on FITC Annexin-V Apoptosis Detection Kit protocol (BD Biosciences, San Jose, CA, USA). Briefly, 3 × 105 cells per well were seeded in a 6-well plate, incubated for one day, and then treated with selected drugs for 48 h. 0.1% DMSO was used as negative control. The cells were detached using trypsin and washed twice with PBS 1X. Then, 1.5 × 105 cells were resuspended in 100 µL of Binding Buffer 1X, stained with FITC-Annexin V and PI, and incubated for 15 min at room temperature in the dark. Finally, the samples were analyzed by FACS Canto flow cytometer and BD Diva Software 6.1.3. Unstained, PI-only, and FITC-only stained samples were used to set the cytometer’s parameters.
7
Cell Cycle Analysis of Reversine and Analogs
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Cell cycle analysis was performed on at least 30,000 events for each sample with FACSCantoTM A using BD Diva Software 6.1.3 (BD Bioscience, San Jose, CA, USA) and the DNA profile was analyzed by ModFit LT 4.1 (Verity Software House, Topsham, ME, USA). Cells were treated with 0.1% DMSO, 1 µM Reversine or different concentrations of each 1 –3 (1 µM, 2.5 µM, 5 µM and 10 µM) for 24 h, detached with trypsin-EDTA, collected by centrifugation and washed three times with PBS. Cells were stained with propidium iodide using BD CycletestTM Plus DNA Kit (BD Bioscience) and then analyzed by flow cytometry. Results obtained are the merge of three experiments.
8
Apoptosis Analysis by Flow Cytometry
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Apoptosis analysis was based on FITC Annexin-V Apoptosis Detection Kit protocol (BD Biosciences, San Jose, CA, USA). Briefly, 3x10 5 cells per well were seeded in a 6-well plate, incubated for one day and then treated with drugs under investigation for 48 hours. 0.1% DMSO was used as negative control. Cells were detached using trypsin and washed twice with PBS 1X. Then, 1.5x10 5 cells were resuspended in 100µL of Binding Buffer 1X, stained with FITC-Annexin V and PI and incubated for 15 minutes at room temperature in the dark. Finally, samples were analyzed by FACSCanto™ flow cytometer and BD Diva Software 6.1.3.
Unstained, PI-only and FITC-only stained samples were used to set the cytometer's parameters.
Unstained, PI-only and FITC-only stained samples were used to set the cytometer's parameters.
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