Cyflow space partec flow cytometer

After isolation, tumors were dissected and digested with 2 mg/mL collagenase D and 0.1 mg/mL DNase I in serum-free RPMI 1640 for 90 min at 37 °C. Tumor-draining axillary lymph nodes (tdLN) were isolated, minced, and filtered to obtain a single-cell suspension. All samples were first preincubated with anti-CD16/32 to avoid nonspecific binding of antibodies to FcγR, and then incubated with a saturating amount of fluorophore-conjugated monoclonal antibodies against CD3ε (145-2C11), CD4 (GK1.5), CD8a (53–6.7), NK1.1 (PK136), F4/80 (BM8), CD40 (1C10), Ly6G/Ly6C (Gr-1; RB6-8C5), CD11b (M1/70), CD25 (PC61.5) and CD206 (MR6F3). For intracellular staining, cells were fixed and permeabilized in accordance with the manufacturer’s instructions (FoxP3/Transcription factor staining buffer set, eBioscence) before staining with fluorophore-labelled antibody against FoxP3 (FJK-16s). Flow cytometric analysis was performed using the CyFlow^® Space Partec flow cytometer (Sysmex, Norderstedt, Germany), and data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

Rhodamine 123 accumulation was analyzed by flow cytometry utilizing the ability of Rhodamine 123 to emit fluorescence. The intensity of the fluorescence is proportional to Rhodamine 123 accumulation. Studies were carried out with Dexverapamil and compounds 1 to 4. NCI-H460 and NCIH460/R cells were grown to 80% confluence in 75 cm² flasks, trypsinized, and resuspended in 10 ml centrifuge tubes in a Rhodamine 123-containing medium. The cells were treated with diterpenes and Dexverapamil (5 and 20 μM) and incubated at 37°C in 5% CO₂ for 30 min. At the end of the incubation period, the cells were pelleted by centrifugation, washed with PBS, and placed in cold PBS. The samples were kept on ice in dark until the analysis of the CyFlow Space Partec flow-cytometer (Sysmex Partec GmbH, Germany). The fluorescence of Rhodamine 123 was assessed on the FL1 channel. A minimum of 20,000 events was assayed for each sample and the obtained results were analyzed using Summit Dako Software.

P-glycoprotein expression level in NCI-H460, NCI-H460/R, DLD1, and DLD1-TxR and cells was measured by flow cytometry. All cell lines were seeded in adherent 6-well plates and treated with Hsp90 inhibitors for 72 h at the following concentrations: 5 µM compound 5, 200 nM compound 7, 100 nM compound 9 (NCI-H460 and DLD1), and 10 µM compound 5, 500 nM compound 7, 500 nM compound 9 (NCI-H460/R and DLD1-TxR). Cells were also treated with 10 nM TQ, a non-competitive P-gp inhibitor, which served as a positive control. In addition, sensitive cells (NCI-H460 and DLD1) were treated with 50 nM DOX and 50 nM PTX that served as positive controls for the induction of P-gp expression. Then, cells were collected by trypsinization, washed with PBS, and directly immunostained by FITC-conjugated anti-P-gp antibody according to the manufacturer’s protocol. The samples were kept in the dark until the analysis on CyFlow Space Partec flow cytometer (Sysmex Partec GmbH, Görlitz, Germany). The fluorescence of FITC-conjugated anti-P-gp was detected on fluorescence channel 1 (FL1-H) at 530 nm. A minimum of 10,000 events were collected for each sample (the gate excluded cell debris and dead cells) and the obtained results were analyzed with Summit software v4.3 (Dako Colorado Inc., Fort Collins, CO, USA).

HEK293T cells were seeded at 1.4×10⁵ cells per well in 24-well plates. 24 h after seeding, cells were co-transfected with 400 ng GoABE or enAsABE expression plasmids (pTK225 or pTK221), 200 ng crRNA expression plasmids (pBD1-2 and pGB125-126), and 400 ng inactive eGFP encoding plasmid (pGB122) obtained from eGFP encoding construct (pRZ174; gift from Toni Cathomen) by QuickChange mutagenesis to introduce premature stop codon, using 2.5 μl TurboFect Transfection Reagent (Thermo Fisher Scientific). The medium was changed at 24 h post-transfection and flow cytometry analysis was performed at 24, 48 and 72 h post-transfection with Partec CyFlow Space flow cytometer (Sysmex Partec, Goerlitz, Germany). Data analysis and visualisation were performed with FlowJo software (FlowJo LLC, Ashland, OR, USA). The initial data of screening has been summarized in Supplementary Table S10. Descriptions and links to plasmid sequences can be found in Supplementary Table S4. Target sequences can be found in Supplementary Table S8.

For detection of reactive oxygen species (ROS) levels in AML cells treated with the peptide, HL-60 and OCI-AML3 cells were incubated with 40 µM CIGB-300 during 30 min, 3 h, and 5 h. Following incubation, cells were collected by centrifugation, washed with PBS, and stained with the fluorescent probe dihydroethidium (DHE) (Sigma, MO, United States) for 30 min at 37°C in the dark. Finally, stained cells were analyzed in the Partec CyFlow Space flow cytometer (Sysmex Partec GmbH, Gorlitz, Germany), and FlowJo software (v7.6.1) (BD, Ashland, OR, USA) was used for data analysis and visualization. In all experiments, 5 mM H₂O₂ and 5 mM N-acetyl cysteine (NAC) anti-oxidant were used as controls.