The cells retrieved from the surface and from the gel were analysed for their content of lymphocyte subsets by flow cytometry (see below). Freshly isolated, non-adherent or the various migrated cells were labelled with a combination of the following antibodies: anti-CD4-PE, anti-CD8-FITC, anti-CD3-PerCP; anti-CD62L-FITC, CD45RA-PE (all from Becton Dickinson, Oxford, UK), anti-CD45RA-CY5 (Serotech, Oxford, UK), anti-CD4-efluor405; anti-CD8a-efluor605 and anti-CD19-PE-Cy7 (all from eBiosciences, Hatfield, UK)

for 30 min on ice. Labelled cells were spiked with a known volume of Flow-Count Fluorospheres (Beckman Coulter, High Wycombe, UK). Cells were counted and their fluorescence analysed using a Cyan flow cytometer and Summit software (both from Dako). In some cases, cells were enumerated by passing buy Ribociclib the entire sample through the flow cytometer. In this way, find more we could separately count and calculate the percentages of the following subsets that adhered, transmigrated or penetrated into the gels: CD4+ or CD8+ T-cells (CD3+), which were of naive (CD45RA +, CD62L +), effector memory (CD45RA −, CD62L −) or central memory (CD45RA −, CD62L +) phenotypes; CD19+ B-cells (Supplemental Fig. 1). Endothelial cells were incubated with non-conjugated

antibodies against E-selectin (1.2B6) or VCAM-1 (1.4C3; both Dako, Ely, UK) for 30 min at 4 °C, washed and incubated with goat anti-mouse FITC-conjugated secondary antibody (Dako) for 30 min

at 4 °C as previously described (McGettrick et al., 2009b and McGettrick et al., 2010). Fibroblasts were incubated with APC-conjugated anti-ICAM-1 (BD Pharmingen, UK) for 20 min at 4 °C. Subsequently, cells were washed and incubated with enzyme-free cell dissociation buffer (Gibco) for 30 min. The dissociated cells were analysed by flow cytometry and C1GALT1 data were expressed as median fluorescent intensity (MFI). Endothelial mRNA was isolated using the RNeasy Mini Kit (Qiagen, Crawley, UK). Gene expression of the chemokines CXCL9, -10, and -11 was analysed by reverse transcription (RT) PCR, followed by densitometry of product bands run on agarose gel containing ethidium bromide, as described (McGettrick et al., 2009b and McGettrick et al., 2010). Data were expressed as a percentage of the β-actin bands. Variation between multiple treatments was evaluated using analysis of variance (ANOVA), followed by comparison of treatments by Bonferroni (inter-treatment) or Dunnett (comparison to control) test as appropriate. Effects of single treatments were analysed by paired or unpaired t-test as appropriate. P < 0.05 was considered as statistically significant. The level of adhesion to EC was slightly higher for cytokine treated than unstimulated cultures, and co-culture with fibroblasts tended to increase this level, but neither effect was statistically significant (Fig. 2A).