Annexin buffer

Cells were stained with fluorochrome-labeled monoclonal antibodies (mAbs): phycoerythrin (PE)/Cy7-conjugated or fluorescein isothiocyanate (FITC)-conjugated anti-rat CD4 (OX-35), PE-conjugated anti-rat CD45RC (OX-22), FITC-conjugated anti-rat TCRα/β (R73), and PE-conjugated anti-rat CD25 (OX-39) from Becton Dickson (BD) Bioscience; Brilliant Violet 510-conjugated anti-rat XCR1 (ZET) and allophycocyanin (APC)-conjugated anti-rat CD103 (OX-62) from BioLegend; eFluor 660-conjugated anti-rat CD62L (OX-85) from Thermo Fisher. When needed, surface markers were stained using appropriate mAbs cocktail prior to fixation and permeabilization with FoxP3/Transcription Factor Staining Buffer Kit according to the manufacturer instructions (eBioscience), using PE-eFluor 610-conjugated anti-FoxP3 (FJK-16 s) from Thermo Fisher. The cells were washed and suspended in PBS-FCS 2%. They were analyzed using a LSRIII Fortessa flow cytometer (BD). Data were analyzed using FlowJo software (TreeStar). A FACSAria instrument (BD) was used for flow cytometry sorting of the CD4⁻ and XCR1⁺ cDC subsets and the CD4⁺ CD62L^high TCRα/β⁺ T cells. For death/apoptosis detection, cells were stained with Violet 450 or APC-conjugated Annexin V in 100 μl of Annexin buffer (BD) and propidium iodide (PI) for 20 min at room temperature in the dark according to the manufacturer protocol (BD Biosciences).

After resuspension of MP pellet in Annexin buffer (BD Biosciences), platelet MPs were labeled with MitoStatus-APC (Thermo-Fisher)/Phalloidin (Sigma-Aldrich, Milan, Italy)/CD41-PerCP-Cy5.5 (BD Biosciences)/AnnexV-V500 (BD Biosciences), as reported in the manufacturer’s instructions, and counted by flow cytometry. MPs were gated based on their size, and the scatter properties were analyzed by running Megamix Plus beads (Biocytex, Marseille, France) at the same photomultiplier (PMT) voltages used for MP detection. Phalloidin negative events (of total MPs or MitoStatus positive MPs) were analyzed for CD41 expression. CD41+ events were then evaluated for their positivity to AnnexinV.

Flow cytometrie (FACS) was performed as follows. After cell treatment with high and low-dose Actinomycin-D and Nutlin-3 for 48 hours, cells were washed with PBS, followed by digestion and mechanical dissociation, as previously described. Enzymatic reaction was stopped with FACS-buffer (10% fetal calf serum solved in PBS). Subsequently, cells were stained with 7-Actinomycin-D and Annexin V (BD Pharmingen, Heidelberg, Germany), both in a 1:10 dilution (7-AAD, Annexin V, FACS buffer) for 20 minutes at 4°C. Staining reaction was stopped with annexin buffer and for the flow cytometric analysis a BD FACS Canto II machine was applied. For analysis, the software FACSDiva (version 6.1.2; Beckton Dickinson) was used.

The induction of apoptosis and necrosis
of U-87 was measured 4 h after X-ray exposure by flow cytometry. X-ray-exposed
unstained and NT-PEO-Por*-stained U-87 were detached from dishes by
0.25% trypsin–EDTA, centrifuged at 1200 rpm for 5 min, and
resuspended in 100 μL of the annexin buffer (BD) and 5 μL
of Annexin V BV421 (BD), which binds to phosphatidylserin (PS) residues
and allows the identification of apoptotic cells. Cells were incubated
a 4 °C for 20 min, washed in the annexin buffer, and centrifuged.
The viability dye 7-aminoactinomycin D (7-AAD, BD) was added to cell
pellets to stain non-viable cells and recognize late-apoptotic fractions.
For fluorescence-activated cell sorting (FACS) characterization, data
were obtained using a FACSAria Fusion cell sorter, equipped with five
lasers, and analyzed with FACSDiva software (ver. 8.0, BD). At least
10 × 10³ events were recorded for each condition.
Debris events were excluded from the analysis by morphological gating
(side scatter vs forward scatter dot plot).

For apoptosis analysis, thymic cell suspensions or suspensions enriched in TECs were washed in Annexin buffer (BD Biosciences) and incubated with AnnexinV (Online Resource 1) plus anti-CD4, anti-CD8, anti-TCRβ or anti-EpCAM, anti-CD45, anti-Ly51 and UEA1-biotin for 20 min. at room temperature in the dark. Detection of UEA1-biotin was performed using a secondary antibody and then resuspended in Annexin buffer. Before analysis, cell suspensions were stained with Sytox Blue Dead Cell Stain (ThermoFisher Scientific). Apoptotic cells were defined as AnnexinV⁺/SytoxBlue⁻ cells. For cell cycle, cells were washed in PBS and incubated in Cytofix/Cytoperm solution (BD Biosciences) at 4 ºC for 30 min. in the dark. After that, cells were washed twice using PermWash buffer solution and incubated in PermWash solution containing 5 μg/mL of Hoechst 33342 (ThermoFisher Scientific) at 4 ºC for 1 hour (h) and 30 min. in the dark. Cycling cells were defined as cells in S + G₂/M cell cycle phase. In both cases, cells were analyzed in a FACS AriaIII cytometer (BD Biosciences) at the Cytometry and Fluorescence Microscopy Centre of the UCM. A minimum of 7 (WT), 6 (DP^hi), 3 (DP^int), 3 (DP^low) mice were used for thymocyte or 6 (WT), 5 (DP^hi), 4 (DP^int), 3 (DP^low) for TEC analysis.