Cell Comb™ Scratch Assay

Protocol tips

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	- NHBE ABI cultures were fixed in 10% neutral buffered formalin 0, 24 h, 48 h, 72 h, and 96 h. After wounding cells are stained with eosin and hematoxylin.

Protocol tips
- NHBE ABI cultures were fixed in 10% neutral buffered formalin 0, 24 h, 48 h, 72 h, and 96 h. After wounding cells are stained with eosin and hematoxylin.

Publication protocol

Mouse and human cells were treated with 2–10 ng/ml recombinant mouse TNF-α (R&D Systems, Minneapolis, MN) or 50 ng/ml human TNF-α (R&D Systems) at 37°C or 39.5°C for up to 6 h or were exposed to a 2-h HS (42°C) coincident with treatment and then incubated for up to a total of 6 h, then analyzed for caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage by immunoblotting. Scratch wounding assays were performed in 28-day NHBE ALI cultures and in confluent A549 and BEAS2B cell monolayers. Because preliminary experiments showed similar mechanical wounding results on uncoated and type I collagen-coated plastic, the remainder of the wounding studies with A549 and BEAS2B cells were performed with monolayers grown on uncoated plastic. Single linear scratch wounds were created in cell monolayers with a 200-μl pipette tip. Wound closure was quantified from daily photomicrographs by measuring the wound area using an overlaid grid and counting the number of boxes devoid of cells. Some of the wounded NHBE ABI cultures were fixed in 10% neutral buffered formalin 0, 24 h, 48 h, 72 h, and 96 h after wounding and embedded on edge in paraffin, and 5-μm-thick cross sections of the cells were placed on positively charged slides and stained with eosin and hematoxylin for photomicrographs as previously described (37). For analysis of gene expression, multiple scratch wounds were made in monolayers cultured in rectangular culture plates using the Cell Comb multiwounding scratch assay kit (Millipore, Billerica, MA).

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