Cellometer auto t4 cell counter

The average cell concentration of 4T1-GL or 4T1-F3 was measured using a benchtop cell counter (Cellometer Auto T4 cell counter, Nexcelom Bioscience, MA). For high number of cells (>150 cells), a 1∶10 serial dilution of various numbers of 4T1-GL (8×10¹–8×10⁵) was spiked into either a volume of 100 µL of pre-warmed culture medium or 100 µL of whole blood freshly harvested from a healthy mouse (n = 4). For low number of cells (0–150 cells), in order to achieve such precision in counting the number of cancer cells spiked into blood samples, we modified our spiking technique from the previous method. Instead of measuring average cell concentration using a benchtop cell counter, we labeled live 4T1-GL or 4T1-F3 cells with non-toxic doses of the very bright fluorescent dye carboxyfluorescein (10 µM, Vybrant CFDA SE Cell Tracer Kit, Invitrogen) in order to manually count the exact number of cells spiked in the sample using a benchtop fluorescence microscope (Fig. S1). Because of the method we used to count the cells, each spiking experiment was unique.

Transfected cells were exposed to 10% Trypan blue solution (Life Technologies, Carlsbad, CA, USA). Total cell number and trypan blue positive cells were determined using the Cellometer Auto T4 cell counter and software according to the manufacturer's instructions (Nexcelom Bioscience LLC, Lawrence, MA, USA).

HT-29 human colon cancer cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and grown in McCoy's 5A medium (Lonza, Walkersville, MD) with 10% FBS (Gemini Bio-products, West Sacramento, CA). A stable clone of HT-29 cells that expresses a scrambled NOX1 shRNA (SC cells), and two independent, clonal HT-29 cell lines that express a NOX1 shRNA producing approximately 65-70% (Si6/G6 cells) or > 90% (6A cells) reduction in NOX1 expression have been described previously [16 (link)]. WiDr, SW403, NCI-H508, and DLD-1 human colon cancer cell lines were also obtained from ATCC and were propagated in RPMI-1640 medium (GE Healthcare Life Sciences, Logan, UT) with 10% FBS. Tumor cells were cultured in a humidified incubator at 37°C in an atmosphere of 5% CO₂ in air. Parental HT-29 tumor cells, and the SC, Si6/G6, and 6A clonal variants were seeded into 60 mm tissue culture plates (Sarstedt, Inc., Newton, NC) at a concentration of 1×10⁵ cells/plate in McCoy's 5A medium containing 10% FBS. After one day in culture, cells adherent to the plates were treated with 50 ng/ml of IL-4 or IL-13; cell proliferation was determined by counting each day using a Cellometer Auto T4 Cell Counter (Nexcelom Bioscience, Lawrence, MA). Every sample was measured in triplicate; the data represent a minimum of three independent experiments.

PMN were isolated from venous blood from consented, healthy, adult volunteers collected into EDTA tubes (Sarstedt Ltd., Leicester, UK). PMN were isolated by overlaying blood on density gradients of histopaque 1077 over histopaque 1119 (Sigma-Aldrich, UK) as described [39 (link)]. Collected PMN were washed using PBSA, counted using a Cellometer auto T4 cell counter (Nexcelom Bioscience Ltd., Manchester, UK), and adjusted to 10⁶ cells/ml in PBSA.
Human umbilical vein endothelial cells (HUVEC) were isolated from umbilical cords by collagenase treatment as described previously [40 (link)]. Umbilical cords were obtained from the Human Biomaterials Resource Centre (University of Birmingham) after informed consent. HUVEC were cultured at 37 °C with 5% CO₂ in M199 supplemented with 20% fetal calf serum, 10-ng/mL epidermal growth factor, 35-μg/mL gentamicin, 1-μg/ml hydrocortisone (Sigma-Aldrich, UK), and 2.5-μg/mL amphotericin B (Life Technologies, CA). Confluent primary HUVEC were detached with trypsin/EDTA (Sigma), washed, and reseeded into 24 well plates (Corning, UK) or into flow channels (Ibidi u-Slide VI (0.4), Thistle Scientific, UK) for studies of vesicle uptake or flow-based adhesion experiments, respectively.

Cells were washed with PBS and trypsinized with 0.25% trypsin for 5 minutes. Cells were diluted in growth medium and mix with equal volume of trypan blue, and 20 ul of mixture was loaded into counting chamber and counted by Cellometer Auto T4 Cell Counter. (Nexcelom Bioscience, Lawrence, MA). All cells were counted 24 hours after administration of the final stress for that condition. For all experiments, cell stress was performed on triplicate cultures (n=3).

Approximately 7.5 × 10⁴ parental or 3′ WRE-Mut cells were seeded in duplicate wells of a 6-well plate. On days one, three, and five after plating, the cells were pelleted, stained with 0.2% trypan blue, and 20 μL of the cell suspension was loaded into a Cellometer cell-counting chamber (Nexcelom Bioscience, CHT4-SD100, Lawrence, MA, USA). Viable cells were quantified using a Cellometer Auto T4 Cell Counter (Nexcelom Bioscience).