Facs fortessa flow cytometer

Activated Tregs were washed three times with PBS and 0.3x10⁶ cells/well were cultured in a 48 well plate for 24 h. Notch1^-/- Tregs infected with pBABE, pBABE NIC-NES and pBABE Grp75 were analyzed as one unit, with data reported in separate figures as indicated in the legends to figures. Cells were harvested and stained with Hoechst 33342 (1 μg/ml), and samples were scored for nuclear damage using fluorescent microscope (Olympus BX-60). Samples were blinded for the experimenter and approximately 200 cells in 5 random fields were scored for apoptotic damage. 0.5x10⁶ activated Tregs were incubated with DiOC₆ (40 nM) diluted in PBS for 10 min at 37°C protected from light. After 10 min, cells were given three washes with pre-warmed (37°C) PBS to remove excess dye and immediately analyzed using BD FACS Fortessa flow cytometer.

HT-29 and SW480 cells were seeded at a density of 30,000cells/cm² in 6-well plates and treated with DOX alone or in combination with fidarestat (30 μM) for 48 h. After treatment, cells were harvested and stained with Annexin V and 7-AAD and incubated on ice for 30 min. The cells were then analyzed using a BD FACS Fortessa Flow Cytometer using a 488 nm laser and detection wavelengths of 530 nm (Alexa Fluor 488) and 670 nm (7-AAD). Data was analyzed using Flow Jo software (Treestar, OR, USA) and gates were drawn using untreated controls.

Cytokines from the isolated and macerated lungs were measured with BD CBA Mouse Th₁/Th₂/Th₁₇ Cytokine Kit and BD CBA Mouse Inflammation kit according to the manufacturer’s instructions (BD Bioscience, San Jose, CA, United States). The Th₁/Th₂/Th₁₇ kit was used for the simultaneous detection of mouse IL-2, IL-4, IL-6, IFN-γ, TNF-α, IL-17A, and IL-10 in a single sample. The Inflammation kit was used for the simultaneous detection of the mouse, IL-6, IL-10, monocyte chemoattractant protein-1 (MCP-1), IFN-γ, TNF-α, and IL-12p70. The kits were used according with the company instructions. Samples were measured on the BD FACS Fortessa Flow Cytometer and analyzed by FCAP Array 3 Software (BD Bioscience). The theoretical limits of detection for the kit Th₁/Th₂/Th₁₇ were 0.1 pg/mL for IL-2, 0.03 pg/mL for IL-4, 1.4 pg/mL for IL-6, 0.5 pg/mL for IFN-γ, 0.9 pg/mL for TNF, 0.8 pg/mL for IL-17A, and 16.8 pg/mL for IL-10 and for the inflammation kit were 5 pg/mL for IL-6, 17.5 pg/mL for IL-10, 52.7 pg/mL for MCP-1, 2.5 pg/mL for IFN-y, 7.3 pg/mL for TNF, 10.7 pg/mL for IL-12p70.

Bone marrow from femurs and tibiae of C57BL/6 mice were obtained according to the protocol established by Inaba et al., (19 (link)). For differentiation in DCs, the cells were cultured in RPMI 1640 medium (LGC, biotechnology, Brazil) supplemented with 10% (v/v) fetal bovine serum (LGC, biotechnology, Brazil) 1% (v/v) penicillin/streptomycin (Gibco, USA), 30 ng/ml of GM-CSF and 15 ng/ml of IL-4 (Invitrogen, USA). The culture medium was changed on the third and fifth days. On day 8, differentiated dendritic cells were obtained. For differentiation in Mφ, the cells were cultured in R20/30 medium (RPMI 1640, 20% (v/v) fetal bovine serum and 30% (v/v) supernatant of L929 cells) (20 (link)). The medium was changed on the fourth day and, on the seventh day, macrophages were obtained. All cells were incubated in a humidified CO₂ incubator at 37°C. The cell phenotype was confirmed by BD FACS Fortessa flow cytometer as described below.

Cells were washed twice with ice-cold PBS and harvested with 200 μL of FACS buffer (1% BSA and 0.5 mM EDTA in PBS) 3 d after transfection. Flow cytometry analysis was performed using a BD FACS Fortessa flow cytometer. A 488-nm diode laser was used for the detection of d2EYFP, a 688-nm diode laser was used for the detection of mCherry, and a 633-nm diode laser was used to detect the iRFP transfection control. In each sample, viable singlet HEK-293T cells were gated via forward-scatter laser and side-scatter and at least 10,000 cells were analyzed as iRFP-positive cells (SI Appendix, Fig. S19). The collected data were analyzed using FlowJo (TreeStar) software. The data represent the results of at least two independent experiments.