Live dead assay

Cell viability of SH-SY5Y and rat DRG cells was assessed using the Live/Dead assay (L3224, Invitrogen). Briefly, cells were incubated for 15 minutes in 10 mL of high glucose DMEM containing 10 μL of 2 mM Ethidium Homodimer-1 (ETH) stock and 20 μL of 4 mM Calcein AM stock in at 37°C protected from light. Following incubation, cell viability was assessed and images captured at 10X magnification using a fluorescent inverted Zeiss microscope (Emission/ excitation; Calcein = 494/517 nm and ETH = 528/617 nm).

A midsagittal cut was made through the explant to examine diffusion through the center of the tissue. The two halves were embedded in O.C.T compound (Tissue Tek), sectioned at 7 μm thickness, fixed in cold acetone, air dried and washed in distilled water. All experimental groups were incubated with streptavidin-horseradish peroxidase (HRP) stain for 30 minutes, washed, counter stained with 3,3′-diaminobenzidine for 10 minutes, washed and finally stained with hematoxylin for 5 minutes. All washes were done with distilled water. The effectiveness of aggrecan depletion was assessed using Safranin O stain and counter stained with Fast Green stain. Tissue sections were deparaffinized in xylene for five minutes followed by a gradual series of ethanol dehydration. The sections were rinsed in distilled water and stained with Fast Green for ten minutes. This was followed by a rinse in 1 % acetic acid and a stain in 0.1 % aqueous Safranin O stain for fifteen minutes. The tissue sections were dried with 100 % ethanol and mounted with synthetic resin. Cell viability after trypsin treatment was assessed using Live-Dead assay (Invitrogen). The slices were dehydrated on clear coverslip and scanned using Aperio ScanScope at 1X magnification using Aperio’s ImageScope Viewer.

Cell viability was assessed using a fluorescent Live/Dead assay (Invitrogen) following standard protocols. Gels were placed in 1.5mL microcentrifuge tubes (Fisher Scientific) and incubated in 1 mM ethidium homodimer and 2 mM calcein AM (Molecular Probes) for 30 min. Fluorescent images were then taken of the entire scaffold using a fluorescence microscope (Axiovert 40 CFL; Zeiss) equipped with a digital camera (11.2 Color Mosaic; Diagnostic Instruments).

A cell viability imaging assay (Invitrogen LIVE/DEAD assay) was used to differentially stain live cells with calcein-AM and dead cells with ethidium homodimer, using protocol guidelines specified by the manufacturer. Cells were imaged using a fluorescent cell imager with 20× magnification (ZOE, Bio-Rad).

Unsorted and sorted (via MACS, FACS, or microchannel) mESCs were analyzed by Live/Dead^® Assay (Invitrogen, Carlsbad, CA), per the manufacturer’s protocol and as previously described(Abruzzese & Fekete, 2013 (link); Holm, 2012 (link); Hwang, Varghese, & Elisseeff, 2007 ; Liu et al., 2010 (link); Liu, Judd, & Lakshmipathy, 2013 (link); Quinlan, 2006 ). Collected cells from each assay were sedimented by centrifugation for 5 minutes at 300 g, washed once with PBS for 5 minutes, spun again for 5 minutes at 300 g, and re-suspended in 1 mL of PBS. A 150 μL aliquot of cells was then allowed to settle briefly on a cover slip. 150 μL of Live/Dead^® reagent (2 μM calcein AM and 4 μM ethidium homodimer 1 in PBS) was added to the cover slip and incubated in the dark for 45 minutes at RT. The cover slip was then flipped onto a glass slide and imaged as previously described. Live (green) and dead (red) cells were counted. Over 2000 cells were counted in each case. For FACS-sorted cells, distinguishing dead cells (ethidium homodimer 1, red) vs. SSEA-1-labeled cells (PE) was readily achieved based on the greater intensity of the fluorescent signal for dead cells, the clear nuclear localization of the signal, and the absence vs. presence of green fluorescence (Figure 4, appendix).