One mL of platelet releasate, washed with 9 mL of Dulbecco’s phosphate-buffered saline (Sigma Aldrich), was centrifuged as described above and the supernatant carefully poured off. The platelet microparticles were resuspended in 400 µL of 0.22 µm filtered Annexin V Binding Buffer (Miltenyi Biotec, Bergisch Gladbach, Germany), and 200 µL of the solution was transferred to a second tube. The solution was then stained with 20 µL of Annexin V FITC (Milteny Biotec), and 2 µL of anti-CD61 APC (clone Y2/51; Miltenyi Biotec), or 22 µL of 0.22 µm filtered Annexin V Binding Buffer for a negative control and incubated for 15 min at room temperature. Finally, 278 µL of 0.22 µm filtered Annexin V Binding Buffer and 50 µL CountBright beads (Thermo Fisher Scientific) were added before analysis. Microparticle gates were set using Megamix-PLUS FSC beads (bead size range: 0.3 to 0.9 µm; BioCytex, Marseille, France), according to our previous report [52 (link)]. At least 2500 bead events were collected. This, and all other flow cytometric analyses, were performed on a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA) using CytExpert ver. 2.4 acquisition and analysis software (Beckman Coulter). An example of gating strategy for PMP quantitation can be found in Figure S1 (see Supplementary Materials ).
Annexin 5 binding buffer
Manufactured by Miltenyi Biotec
Sourced in Germany
Annexin V Binding Buffer is a buffer solution used to facilitate the binding of Annexin V to phosphatidylserine residues on the cell surface. Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine. The buffer provides the appropriate ionic conditions and pH to enable this binding interaction.
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Lab products found in correlation
Annexin 5 fitc, by Miltenyi Biotec (3 mentions)
Propidium iodide, by Miltenyi Biotec (3 mentions)
Apc conjugated annexin 5, by BioLegend (2 mentions)
Macsquant analyzer 10, by Miltenyi Biotec (2 mentions)
Rnase a, by Qiagen (2 mentions)
Countbright beads, by Thermo Fisher Scientific (1 mentions)
Megamix plus fsc beads, by Biocytex (1 mentions)
Annexin 5 binding buffer, by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline, by Merck Group (1 mentions)
Cytoflex, by Beckman Coulter (1 mentions)
Macsquant analyser, by Miltenyi Biotec (1 mentions)
Annexin 5 binding buffer, by Merck Group (1 mentions)
Flowlogic software, by Miltenyi Biotec (1 mentions)
Fitc mouse anti human cd62p, by BD (1 mentions)
Cd62p fitc, by BD (1 mentions)
Mouse igg1 fitc, by BD (1 mentions)
Facsflow, by BD (1 mentions)
Igg1 fitc, by Miltenyi Biotec (1 mentions)
Facscan, by BD (1 mentions)
7 aad, by Miltenyi Biotec (1 mentions)
12 protocols using annexin 5 binding buffer
1
Platelet Microparticle Quantitation by Flow Cytometry
2
Comprehensive Cell Cycle and Apoptosis Analysis
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Flow cytometry analyses of apoptosis rates and cell cycle distribution were performed by AnnexinV/propidium iodide (PI) or PI-staining, respectively. Briefly, for PI staining, cells were washed once with PBS and fixed with 70% methanol dissolved in PBS. After washing, cells were re-suspended in staining buffer, containing 2 μg/mL PI (Sigma-Aldrich) and 100 μg/mL RNAse A (Qiagen, Hilden, Germany) dissolved in PBS before measurement. For apoptosis measurement, cells were washed with Annexin V-binding buffer (Miltenyi, Bergisch Gladbach, Germany) before incubation with 2.5 μL Annexin V-FITC (Miltenyi) and 7.5 μL PI (2 mg/mL stock, Sigma-Aldrich) dissolved in 70 µL Annexin V-binding buffer for 15 min at room temperature (RT) in the dark. Afterwards, samples were re-suspended in 500 µL Annexin V-binding buffer. Measurement of cell proliferation was performed using the EdU Flow Cytometry Kit detecting Eterneon-Red 645 Azide according to the manufacturer’s protocol (Baseclick, Neuried, Germany). A flow cytometric analysis was performed by measuring at least 5 × 104 cells using the MACSQuant Analyser and evaluated by the Flowlogic software (both Miltenyi Biotech).
3
Platelet Characterization and Apoptosis
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Immediately after isolation, 2 µl mouse platelets were incubated with antibodies against IgG1-FITC (Milteny Biotec, Bergisch Gladbach, Germany, order no.: 130-098-847, 1:25) or CD62P-FITC (BD Biosciences, Heidelberg, Germany, Cat: 561923, 1:25). Human platelets were incubated with antibodies against FITC Mouse IgG1 (BD Biosciences, Cat: 555748, 1:25) or FITC Mouse Anti-Human CD62P (BD Biosciences, Cat: 555523, 1:25). Subsequently, 46 µl of FACs buffer (2 mM EDTA, 0.5 % FCS ad 100 ml PBS, pH 7.1) was added and the samples incubated for 30 min at 4 °C in dark. To study apoptosis, 2 µl of platelets were incubated with 5 µl Annexin V-FITC (Milteny Biotec, Order no.: 130-097-928) in 100 µl Annexin V Binding Buffer (Miltenyi Biotec, 20x Stock Solution) at RT for 30 min in dark. Immediately before analysis, 2 µl of 7-AAD (Miltenyi Biotec) was added. All samples were centrifuged at 300g for 10 min and the pellets were resuspended in 100 µl of FACs flow (BD FACSFlow). FACs analysis was instantly performed with a BD FACScan.
4
Annexin-V and 7-AAD Apoptosis Assay
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To assess early apoptosis, cells were stained with Annexin-V fluorescein isothiocyanate (FITC) and 7-amino-actinomycin D (7-AAD; BD Via-Probe) in Annexin-V binding buffer (Miltenyi Biotec, Bergisch Gladbach, Germany). From each sample, approximately 1 × 105 cells were processed. All of the samples were measured using a BD FACS Canto II flow cytometer (Becton-Dickinson). BD FACSDiva (Becton-Dickinson) software was used for the data analysis.
5
Apoptosis and Necrosis Analysis
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Viability analysis was done using APC-conjugated Annexin V (Biolegend Cat. No. 640920 and propidium iodide (Miltenyi Biotec Cat. No. 130–093-233). Briefly, adhered RH30 cells following trypsinization are transferred into a U-bottomed 96-well plate for staining. Cells are stained for 20 min at room temperature with APC-conjugated Annexin V (Biolegend cat # 640,920; 1:100 dilution) and propidium iodide (Miltenyi Biotec cat# 130–093-233; 1:100 dilution) diluted in Annexin V Binding Buffer (Cat. No. 422201) followed by 2X washes in warm Annexin V Binding Buffer and analysis by flow cytometry.
6
Apoptosis Assay of PBMCs
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PBMCs were cultured with various concentrations of daunorubicin, tacrolimus, MPA and prednisolone (Sigma) and apoptosis detected by intracellular staining for activated caspase 3 (BD Pharmingen, Wokingham, UK) or by annexin V (Miltenyi Biotec) surface staining after 24 or 48 h, respectively. annexin V staining was performed for 15 min at room temperature in annexin V binding buffer (Miltenyi Biotec); 50 M Verapamil (Sigma) was added at the beginning of culture. Percentages expressing annexin V at each concentration were background subtracted for annexin V expression in media alone.
7
Comprehensive Immune Profiling of Cohorts
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MLs phenotypes from patients’ samples of the ELYP cohort at different timepoints were performed using CD103-FITC, CD73-PE, CTLA4-APC, CD39-APC-Vio770, CCR6-PE, PD1-PE-Vio770, NKG2D-APC, CD45RO-APC-Vio770 (Miltenyi), CD3-AF700, CD161-PE-Cy5, CD8-BV605 and CD4-BV711 (BD Biosciences), . MLs phenotypes from patients’ samples of the REMIND and IMCO cohort at different timepoints were performed using CD69-FITC, CD45RO-PerCP, CD5-AF700, CD8-BV605, CD4-BV711 (BD Biosciences), NKG2D-APC and CD103-APC-Vio770 (Miltenyi). All samples were co-stained with DAPI to assess cell viability and diluted in optimal concentration in FACS buffer (Miltenyi).
For the allogenic cocultures, cells were then stained using the following antibodies: Annexin V-FITC, E-Cadherin-PE-Vio770 HLA-E-APC, MICA/MICB-APC-Vio770 (Miltenyi), HLA-ABC-AF700 (BioLegend), HLA-DPDQDR-BV510, CD45-BV605 (BD Biosciences) and ULBP 2/5/6-PE (RD). All samples were co-stained with DAPI to assess cell viability and diluted in optimal concentration in Annexin V binding buffer (Miltenyi).
For the allogenic cocultures, cells were then stained using the following antibodies: Annexin V-FITC, E-Cadherin-PE-Vio770 HLA-E-APC, MICA/MICB-APC-Vio770 (Miltenyi), HLA-ABC-AF700 (BioLegend), HLA-DPDQDR-BV510, CD45-BV605 (BD Biosciences) and ULBP 2/5/6-PE (RD). All samples were co-stained with DAPI to assess cell viability and diluted in optimal concentration in Annexin V binding buffer (Miltenyi).
8
Apoptosis Assay of HepG2 Cells
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PBS was used to wash the cells from each group, then it was harvested using trypsin EDTA. Then it was centrifuged and counted using hemacytometer. To 5 × 105 accounted cells, Annexin V binding buffer (130-092-820, Miltenyi Biotec, Bergisch Gladbach, Germany) as much as 500 μL, Anti-FITC (130-048-701, Miltenyi Biotec, Bergisch Gladbach, Germany) as much as 5 μL, and Propidium Iodide (130-093-233, Miltenyi Biotec, Bergisch Gladbach, Germany) as much as 5 μL were added and continued by incubation in the dark at 4 °C. The HepG2 cells apoptotic, necrotic, death, and live cells percentage were measured by MACSquant Analyzer 10 (Miltenyi Biotec, Bergisch Gladbach, Germany) (Widowati et al., 2018a ; Widowati et al., 2020 ).
9
Apoptosis and Necrosis Analysis
10
MAIT Cell Assays with E. coli-Stimulated THP1
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THP1 cells (ECACC, UK) were incubated overnight with paraformaldehyde (PFA)-fixed Escherichia coli (E. coli; DH5α, Invitrogen) at a ratio of 25 bacteria per cell, or a sterility control. THP1 cells were washed extensively, and PBMCs, intrahepatic lymphocytes, enriched CD8+ T cells, or sorted CD161++ Vα7.2+ T cells were added to the THP1 cells for a 5-h stimulation. Brefeldin A (eBioscience) was added for the final 4 h of the stimulation. Alternatively, for the assessment of degranulation, anti-CD107a-PE-Cy7 (BioLegend) was added from the start of the stimulation. For the assessment of GrB and perforin upregulation, PBMCs were added to E. coli-treated THP1 cells for 24 h. MAIT cell apoptosis was detected by Annexin V surface staining (Miltenyi Biotec), in Annexin V binding buffer (Miltenyi Biotec) for 15 min. Staurosporine (Sigma Aldrich) was added as a positive control. Alternatively, MAIT cells were stimulated with IL-12+ IL-18 (both Miltenyi Biotech) at 50 ng/ml for 20 h, and Brefeldin A (eBioscience) was added at 3 µg/ml for the final 4 h of the incubation. For blocking experiments, anti-MR1 antibody (gift from Professor Ted Hansen or from Biolegend), anti-IL-12p40/70, anti-IL-18 antibody (both Miltenyi Biotech), or the appropriate isotype controls were added at 10 µg/ml. Anti-CD8α antibody (clone LT8; Novus Biologicals) was added at the indicated concentrations.
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