Novocyte 2000 flow cytometer

NIH/3T3 or RAW264.7 cells cultured in 24-well plates (Thermo Fisher Scientific) were harvested and collected by centrifugation at 350× g and 4 °C for 5 min. The RAW264.7 cells were treated with TruStain FcX (anti-mouse CD16/32 antibody, clone: 93, BioLegend, San Diego, CA, USA) for 15 min at 4 °C to block FCγ receptors III and II. Next, the cells were stained with APC-labeled anti-mouse MMR (APC-MMR) antibody (clone: C068C2, Biolegend) or APC-labeled mouse IgG2a,κ isotype control antibodies (clone: MOPC-173, Biolegend) for 10 min at 4 °C. The cells were then collected by centrifugation at 350× g and 4 °C for 5 min, washed with phosphate buffered saline containing 0.1% bovine serum albumin, and passed through a 35-μm cell strainer (Corning, Corning, NY, USA). The fluorescence intensity of the cells was measured using a NovoCyte 2000 flow cytometer (ACEA Biosciences, San Diego, CA, USA). The APC-MMR antibody-stained cells were excited using a 640-nm laser and detected using a 675 ± 30-nm bandpass filter. In all, 10,000 cells were counted for each sample, and the fluorescence intensity of the cell population was determined using Novo Express software (version 1.2.5, ACEA Biosciences).

Cells were stained with allophycocyanin (APC)-labeled rat anti-mouse MSR-A (CD204) antibody (APC-anti-MSR-A; clone: M204PA; Thermo Fisher Scientific, Waltham, MA, USA) or APC-labeled rat IgG2a,κ isotype control antibody (clone: eBR2a; Thermo Fisher Scientific) for 30 min on ice. The Fc receptors in RAW 264.7 cells were blocked with TruStain FcX (anti-mouse CD16/32; clone:93; BioLegend, San Diego, CA, USA) for 30 min on ice before the cells were labeled with antibodies. The stained cells were subsequently collected through centrifugation at 200 × g at 4°C for 3 min, washed twice with PBS containing 0.1% BSA, and passed through a 35-μm cell strainer (Corning, Corning, NY, USA). The fluorescence intensity of the cells was measured using a NovoCyte 2000 flow cytometer (ACEA Biosciences, San Diego, CA, USA). The stained cells were excited using a 640-nm laser and detected using a 675 ± 30-nm bandpass filter. A total of 10,000 cells were counted for each sample, and the fluorescence intensity of the cell population was determined using the Novo Express software (version 1.2.5, ACEA Biosciences).

Tumors were harvested from mice and single cell suspensions were made by incubating minced tumor at room temperature for 1 h in enzyme digestion buffer containing type V collagenase, type IV DNase, and type V Hyaluronidase. Cells were washed and incubated for 30 min on ice in staining buffer (Thermo Fisher Scientific, Waltham, MA, USA) with different combinations of the following antibodies: FITC-conjugated anti-CD4 (RM4-5), anti-CD8a (53-6.7), anti-Gr-1 (RB6-8C5), PE-conjugated anti-49b (DX5), anti-CD8a (53-6.7), PerCP-Cy5.5-conjugated anti-CD45 (30-F11), APC-conjugated anti-CD3 (17A2), and anti-CD11b (M1/70) antibodies (Thermo Fisher Scientific, Waltham, MA, USA). For intracellular staining of FoxP3, cells pre-stained with antibodies targeting surface proteins were incubated with 1× fixation/permeabilization buffer and subsequently stained with PE-conjugated anti-FxoP3 (FJK-16s) antibody in 1× permeabilization buffer (Thermo Fisher Scientific, Waltham, MA, USA). FACS analysis was performed on a NovoCyte 2000 Flow Cytometer using NovoExpress software (ACEA Biosciences, San Diego, CA, USA).

A549 cells were plated in 6 cm plates and allowed to attach for 24 h and then treated with or without essential oils for 48 h. Only adherent cells were harvested and then washed twice with ice-cold PBS. After fixed gently in ice-cold 70% EtOH and incubated for 2 h at −4°C, cells were washed with ice-cold PBS twice. Then, fixed cells were incubated with 1 mg/mL propidium iodide (PI) (Invitrogen, USA) in PBS at room temperature for 30 min. Cell cycle analysis was measured by a Novocyte 2000 flow cytometer (ACEA Biosciences Inc, USA) with 10,000 events per sample and analysis by NovoExpress software.