Recombinant osteopontin

Mouse brain microvascular endothelial cells (MBMEC) at 3 days post isolation were seeded in 24-well plates at passage 1. These cells were treated with recombinant osteopontin (R&D Systems) with or without anti-osteopontin IgG (R&D systems) at the indicated concentrations at 48 h post plating when the cells reached confluency. The treatment period was for 24 h followed by harvesting the cells to obtain RNA and cDNA exactly as described previously^{12 (link)} using RNeasy micro kit (Qiagen) followed by cDNA synthesis (Revertaid kit, Thermofisher). qRT-PCR was performed using Absolute qPCR SYBR Green Fluorescein Mix in IQ5 instrument (Biorad) using a previously established PCR protocol^{12 (link),32 (link),38 (link)}. Rplp0 was used as a house-keeping gene and qRT-PCR was performed for Spp1, Cd44 and Cdh5 using 2^−ΔCt method. Primers are listed in Supplementary Table S3.

Enriched CD45⁺ IEL (1x10⁵ cells/well) from Rag-2^-/- or E8_I^-/-Rag-2^-/- mice were cultured in a 96-well flat-bottomed well plate in RPMI complemented with 10% fetal bovine serum, penicillin/streptomycin, HEPES, L-glutamine and β-mercaptoethanol in the presence or absence of 2 μg/ml of recombinant osteopontin (R&D) for 4 hours. After incubation, cells were recovered and stained for surface markers, 7AAD and annexin V. In other experiments, enriched CD45⁺ IEL (1x10⁵ cells/well) from Spp-1^-/-Rag-2^-/- mice were cultured in the presence of enriched iCD8α cells (1x10⁵ cells/well) from Rag-2^-/- mice for 4 hours. After incubation, cells were recovered and stained for surface markers, 7AAD and annexin V. In other experiments, iCD8α cell-depleted CD45⁺ IEL (1x10⁵ cells/well) from Spp-1^-/-Rag-2^-/- mice were incubated in the presence or absence of enriched iCD8α cells (1x10⁵ cells/well) from Rag-2^-/- or Spp-1^-/-Rag-2^-/- mice for 4 hours. After incubation, cells were recovered and stained for surface markers, 7AAD and annexin V. For all in vitro survival experiments, IEL were gated according to their size in a forward versus side scatter plot without prior exclusion of dead cells. Then, cells were selected by their expression of NKp46 and NK1.1 followed by analysis of 7AAD incorporation and annexin V staining.