100mm tissue culture plates

Human foreskin keratinocytes (HFKs) were prepared from human foreskins donated by Georgetown University Hospital. HFKs were maintained in Keratinocyte Serum Free Media (Invitrogen, Carlsbad, CA), supplemented with 50 g/ml bovine pituitary hormone, 26 ng/ml recombinant epidermal growth factor, and 10 g/ml gentamycin (KGM). All cells were maintained using T75 or T175 flasks. RNA or western blot lysates were collected from 100mm tissue culture plates, all from BD Falcon (San Jose, CA).

MDA-MB-231 cells (0.75–2 × 10⁶) were plated in 100-mm tissue culture plates (BD Falcon) and allowed to adhere overnight. Cells were then treated with P3A1-MWCNT[1], P3A1, DSPE-mPEG coated MWCNT, P3A1-DSPE-mPEG, DSPE-mPEG, DSPE-mPEG coated MWCNT, P3A1-DSPE-mPEG, or saline at the specified concentrations for 48 and 72 hours. Cells were washed with PBS and co-stained with Annexin V (APC) and propidium iodide (BD Pharmingen) per the manufacturer’s protocol. Briefly, cells were trypsinized, pelleted, washed twice with cold PBS, and then suspended in 1 × Annexin V binding buffer at a concentration of 1 × 10⁶/mL. 1 × 10⁵ cells were then mixed with Annexin V and incubated for 15 minutes at room temperature in the dark then 400 uL of 1 × Binding Buffer with or without propidium iodide (2.0 µg/mL) was added, mixed, and samples were analyzed on the Accuri6 Flow Cytometer (BD Biosciences). Analysis of data was performed using FCS Express version 3 (De Novo Software). For cell cycle analysis, cells were treated as indicated, fixed in 50% ice-cold ethanol, washed once in PBS, and then were treated with FxCycle PI/RNase staining solution (Life Technologies) per the manufacturer’s protocol. Analysis was performed using ModFit software.

GBM or NHA cells were plated in 100 mm tissue culture plates (BD Falcon) and allowed to recover for 1–4 days. Plates were treated with 2% DSPE-PEG MWCNTs at the specified concentrations for 24 hours. Cells were washed with PBS and co-stained with Annexin V (APC) and propidium iodide (BD Pharmingen) per the manufacturer’s protocol. Briefly, cells were trypsinized, pelleted, washed twice with cold PBS, and then suspended in 1X Annexin V binding buffer at a concentration of 1×10⁶ cells/ml. 1×10⁵ cells were then mixed with Annexin V and incubated for 15 minutes at room temperature in the dark. Next, 400 ul of 1X Binding Buffer was added, mixed, and samples were analyzed on the Accuri6 Flow Cytometer (BD Biosciences). Analysis of data was performed using FCS Express version 3 (De Novo Software).