Sybr green detection

To assess proliferation, tumor cell-seeded scaffolds were lysed in Caron’s buffer and sonicated at low power to liberate cellular DNA. Total DNA content was measured from these samples by the QuantiFluor® dsDNA System (Promega) according to manufacturer instructions. To quantify secretion of IL-8, tumor cell-seeded scaffolds were cultured in low serum prior to collection of media, which was then used in the IL-8 ELISA (R&D Systems) according to manufacturer instructions. To analyze IL-8 gene expression, total RNA was harvested from tumor cell-seeded scaffolds with TRIzol® (Invitrogen) according to manufacturer instructions and 1 μg was reverse transcribed to cDNA (qScript cDNA supermix, Quanta BioSciences). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR green detection (Quanta BioSciences) on an Applied Biosystems 7500 System. The following primer sequences were used: human IL-8 (fwd: 5’- agaaaccaccggaaggaaccatct-3’, rev: 5’- agagctgcagaaatcaggaaggct-3’) and human β-actin (fwd: 5’ - aatgtggccgaggactttgattgc-3’, rev: 5’- aggatggcaagggacttcctgtaa-3’) (IDT Technologies). Quantitative analysis was performed based on the ΔΔC_t method [38 (link)]. Sample size was n = 3 or greater per condition.

Secreted protein levels were measured by ELISA (R&D Systems) following 24 hours of culture in serum-free media, according to the manufacturer instructions. Cells were released by dissolving alginate disks with ethylenediaminetetraacetic acid (EDTA) and lysed in Caron’s buffer. DNA content of cell lysates was fluormetrically measured with Quant-iT PicoGreen dsDNA reagent (Invitrogen) and used to normalize secretion levels. To determine changes at the transcriptional level, total RNA was harvested from isolated cells using TRIzol. Reverse-transcription of 1 μg to cDNA (qScript cDNA supermix, Quanta BioSciences, Gaithersburg, MD) was performed with random hexamer and IL-8 specific oligo(dt) primers, followed by qRT-PCR using SYBR green detection (Quanta Biosciences) and an Applied Biosystems 7500 system. Quantification was normalized relative to β-actin using the ΔΔC_t method.