Accury c6 flow cytometer

Propidium iodide (PI) incorporation was performed as previously described^{37 (link)}. Briefly, shIBTKα-transduced and shCTRL-transduced DeFew cells (3 × 10⁵) were centrifuged, and pellets were suspended in fluorochrome solution (0.1% sodium citrate, 0.1% Triton X-100, 50 mg/l PI, in deionized water). After 60 min-incubation at 4 °C, cells were analysed using Accury C6 Flow cytometer (Becton Dickinson).

Cell death was measured using 1 µg/mL APC Annexin V (BD Pharmingen, San Jose, CA) and 0.5 µg/mL propidium iodide (PI) (MERCK). In brief, 5 × 10⁴ cells/well in 24-well dishes (Life Sciences) were incubated in the presence or absence of different concentrations of IMMUNEPOTENT CRP (ICRP), epirubicin (EPI, Sigma-Aldrich, ST. Louis, MO) or cyclophosphamide (CPA, Sigma-Aldrich) for 24 h in complete DMEM or RPMI to a final volume of 400 μL to obtain the ICRP CC₅₀. For cell death inhibition assays, cells were pre-treated 30 min with or without 5mM N-acetyl-L-cysteine (NAC), 10 μM QVD.oph, 30 μM Necrostatin-1 (NEC-1) or 15 μM Spautin-1 (Sp-1), and then co-treated with or without ICRP CC₅₀ for 24 h, as mentioned before. Cells were then detached, washed twice with PBS, suspended in 100 μL of binding buffer (10 mM HEPES/NaOH, pH 7.4, 140 mM NaCl and 2.5 mM CaCl₂), stained and assessed by BD Accury C6 flow cytometer (Becton Dickinson, Franklin Lakes, NJ). The results were analysed using FlowJo Software (LLC, Ashland, OR).

Cell death quantification was determined by analyzing phosphatidylserine exposure using annexin V-allophycocyanin (APC) (AnnV, 0.25 μg/mL; BD Biosciences Pharmingen, San Jose, CA, USA) and cell membrane permeability with propidium iodide (PI; 0.5 μg/mL; MilliporeSigma, Eugene, OR, USA) staining. In brief, 5×10⁴ cells were seeded and exposed to different concentrations of ICRP in subsequent assays; this allowed to define the median cytotoxic concentration of ICRP required to induce 50 % of cell death (CC₅₀). After 24 h of treatment, cells were recollected and washed with phosphate-buffered saline (PBS), then resuspended in binding buffer (10 mM HEPES/ NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2), and stained during 20 minutes at 4 °C. Finally, cells were assessed in BD Accury C6 flow cytometer (Becton Dickinson, Franklin Lakes, NJ) and analyzed in FlowJo Software (LLC, Ashland, OR).

Cell cycle analysis was performed as described in [50 (link)]. Briefly, cells were washed in PBS, fixed with 70% ice-cold ethanol, and incubated at −20 °C overnight. Fixed cells were washed in PBS and stained with a solution of propidium iodide 5 μg/mL (Sigma-Aldrich, Saint Louis, MO) and RNAse 20 μg/mL (Sigma-Aldrich, Saint Louis, MO, USA) in PBS. Samples were incubated overnight at 4 °C and DNA content of at least 20,000 cells was evaluated with a BD AccuryC6 Flow Cytometer (Beckton Dickinson, Franklin Lake, NJ, USA). Data were analyzed with R-BiocManager software (flowCore/flowViz package).

The NCI-H929 cells (1 × 10⁶ cells/well) were treated with secalonic acid for 24 h and 48 h (in humidified 5% CO₂ atmosphere at 37 °C), and apoptosis/necrosis was assessed by the flouresceinisothiocynate (FITC)-conjugated annexin V/PI assay kit (eBioscience™Annexin V; Invitrogen, Germany) by flow cytometry. Briefly, treated cells were centrifuged at 1200 rpm for 5 min and washed twice with ice-cold PBS (phosphate-buffered saline) and 1 × binding buffer, respectively. After the cells were re-suspended in binding buffer at a total number of 4 × 10⁶ cells/mL, 5 µL of FITC-conjugated annexin V was added to 100 µL of the cell suspension and incubated for 15 min at room temperature. Then, the cells were washed with 1 × binding buffer and resuspended in 200 µL of 1 × binding buffer. Propidium iodide staining solution (5 µL) was added and analyzed after 15 min incubation at room temperature in the dark using BD Accury C6 Flow Cytometer (BD Biosciences). Apoptosis and necrosis were evaluated on fluorescence 2 (FL3 for propidium iodide) versus fluorescence 1 (FL1 for annexin) plots. The fluorescent cells % in each quadrant pointed out different cell populations corresponding to viable and non-apoptotic (annexin V−PI−), early apoptotic (annexin V+PI−), late apoptotic as well as early necrotic (annexin V+PI+) and late necrotic (annexin V−PI+) cells.

Tumours, TDLN and spleens were obtained from mice 28 days post-tumour inoculation (p.i.). Lymphoid organs were disrupted through a 1-mm metal mesh and seeded at a final concentration of 1 × 10⁷ cells/ml in complete RPMI medium. Tumours were cut in small pieces and equal quantities of tissue were incubated in complete RPMI medium. After 24 h, the conditioned medium was obtained. Mice interferon (IFN)-γ (BDB-558473), tumour necrosis factor (TNF)-α (BDB-558480) and IL-10 (BDB-558300) CBA Flex Sets (BD Biosciences) were used to analyse specific cytokines in a BD Accury C6 flow cytometer, following the manufacturers’ instructions. Results were analysed with FCAP Array Software v3.0 (BDB).

Single-cell suspensions obtained from tumours, tumour draining (TDLN) and non-draining lymph nodes and spleens were stained with antibodies against various cell surface markers using standard staining methods, as previously described.^{22 ,23 (link)} The following panel of commercially available and fluorochrome conjugated anti-mouse monoclonal antibodies were used in the study: FITC-anti-mouse CD3 (BDB-561798), FITC-anti-mouse CD4 (BDB-557307), PE-anti-mouse CD4 (BDB-557307), PE-anti-mouse CD8 (BDB-553032), PE-anti-mouse CD19 (BDB-557399), FITC-anti-mouse Gr1 (BDB-553126), PE-anti-mouse CD11b (BDB-557397), FITC-anti-mouse CD49 (BDB-561066), FITC-anti-mouse CD44 (BDB-561859) and APC-anti-mouse CD25 (BDB-558643), all purchased from BD Biosciences (BDB; San José, CA, USA). Samples were run on a BD Accury C6 flow cytometer (BDB) and data were analysed using the BD Accury C6 software (BDB).