Phc3016

Here, 5 × 10⁵ BV-2 cells were seeded into six-well plates, and 5 × 10³ were seeded onto a Millicell EZ slide (Millipore) in LCM and grown to 80% confluency. Cells were treated with CXCR5 antibody low endotoxin, sodium azide-free (ab225575, 1:100, Abcam). Mouse IgG (ab37355, 1:100, Abcam) was used as a control. Following a 6-h incubation, the LCM medium was removed, and the cells were washed with PBS, and fresh FBS free DMEM media was supplied. Cell cultures were then stimulated with 10 ng/ml of recombinant mouse interleukin-4 rmIL-4 (PMC0045 Thermo Fisher Scientific), and the other wells were treated with 20 ng/ml of recombinant mouse TNF-α (rmTNF-α, PHC3016, Thermo Fisher Scientific) and supplemented with 10 ng/ml of recombinant mouse interferon gamma (rmIFNγ, PMC4034, PHC3016, Thermo Fisher Scientific). Following 12 h of incubation, the cells were washed with PBS, and the total RNA was extracted and purified (RNeasy Purification Kits; Qiagen) using RT-PCR analysis.
BV-2 cells that were seeded into Millicell EZ slides were washed with PBS and fixed with 2% formaldehyde (Sigma-Aldrich) for 10 min for immunocytochemistry analysis.

Bovine caudal IVDs were harvested retaining superior & inferior vertebral endplates from bovine tails obtained from a local abattoir (Green Village Packing Co., NJ). Following isolation, endplates were cleaned with a wound debridement system (Pulsavac, Zimmer, Warsaw, IN) to remove potential blood clots and rinsed with 70% ethanol and washing solution (3% penicillin/streptomycin and 1.5% fungizone in PBS). All IVDs were cultured at 37°C and 5% CO₂. For all studies, control culture medium consisted of high glucose DMEM, 10% FBS, 50ug/mL ascorbic acid, 1% penicillin/streptomycin, 0.5% fungizone (Fisher-Scientific, Waltham MA), and 1:500 primocin (Invivogen, San Diego, CA) and all TNFα groups were cultured in control medium + 100ng/mL human recombinant TNFα (Invitrogen PHC3016). All reagents were obtained from Invitrogen (Carlsbad, CA) unless otherwise noted. TNFα was used because it is typically expressed following tissue injury [17 (link)], is associated with chronic painful conditions of the spine, and is considered an initiator of a larger pro-inflammatory and catabolic cascade in the IVD [31 (link)]. TNFα can also be interpreted as a model pro-inflammatory cytokine since it is similar in size to other pro-inflammatory cytokines known to be important in IVD degeneration, such as IL-1β and IL-6.