Cytocount beads

To assess the viability over prolonged culture of HSPC-pDCs or blood pDCs, cells were collected at each time point and subsequently analyzed using flow cytometry. Cells were stained with Zombie aqua as a viability dye, and viable cell counts were measured and normalized to a fixed number of counting beads (CytoCount beads; Dako).

CD4 T cell-derived EVs and CEM-EVs were analyzed using high-sensitivity flow cytometry as previously described^{49 (link)}. Thirty microliters of EV samples was incubated with the appropriate amount of specific antibodies and 10 μL of FITC-bound Annexin V reagent (Tau Technologies, Netherlands). Each stained sample was analyzed on a 3-laser Navios flow cytometer (Beckman-Coulter), based on a protocol standardized with Megamix-Plus FSC beads (BioCytex, Marseille, France). The following fluorescently-tagged antibodies were procured from Beckman Coulter: PE-tagged anti-TCRαβ, APC-tagged anti-CD45, PE-tagged anti-CD3, and PE-tagged anti-CD4 and their respective controls. The presence of EVs derived from different cellular origins was determined using PC7-tagged anti-CD41, APC-tagged anti-CD11b, Alexa 750 APC-tagged anti-CD235, PE-tagged anti-CD31 and their respective control antibodies. The presence of apoptotic bodies was determined by colabeling with FITC-bound Annexin V and DAPI (4′,6′-diamidino-2-phenylindole). Before analysis, CytoCount beads (Dako, Copenhagen, Denmark) were added as internal standards to samples to determine the concentration of EVs. Flow cytometry data were analyzed using Kaluza 1.5a Software (Beckman Coulter).

A short-term homing assay in which the recruitment of L-selectin expressing, and L-selectin deficient T cells are compared directly in individual mice was used as described previously (11 (link)). Briefly, Thy1.1 mice were subcutaneously injected with 1.5 × 10⁶ NP68-B16 tumor cells and tumors grown for 7–14 days. Thy1.1 mice served as non-tumor bearing mice. Tumor bearing mice and non-tumor bearing mice received 597cGy TBI. CD8⁺ T cells isolated from naïve F5LΔP and F5LselKO (both Thy1.2) were mixed 1:1 (numbers determined by haemocytometer, and confirmed by flow cytometry using Cytocount beads) and a total of 6–10 × 10⁶ T cells injected intravenously into mice at 14.00–16.00 GMT on the day following irradiation. All mice received 100 μg NP68-IFA s.c. prior to T cells. The next morning (10.00–11.00 GMT; 18 h following T cell injections), mice were culled and tissues including blood, spleen, tumor-draining and peptide-draining lymph nodes, non-draining lymph nodes and tumor were collected. Tumors were disaggregated using gentle MACS (Miltenyi). Lymphoprep was used to isolate immune cells from blood and tumor by centrifugation. Following isolation, cells were stained for CD8, Thy1.2, and L-selectin, analyzed by flow cytometry and L-selectin expression on donor CD8⁺, Thy1.2 cells determined. Cell numbers were determined using Cytocount beads (Dako).

293Ts were harvested with enzyme-free cell dissociation buffer and Dynabeads Human T-Activator CD3/CD28 beads were removed from primary human CD4+ T cells using a DynaMag-2 magnet (Invitrogen). Typically 2×10⁵ washed cells were incubated for 30 mins in 100 µL PBS with the indicated fluorochrome-conjugated antibody or streptavidin-APC. All steps were performed on ice or at 4°C and stained cells were analysed immediately or fixed in PBS/1% paraformaldehyde. Viability was assessed using forward and side scatter, and absolute cell numbers determined using CytoCount beads (Dako) as a reference population according to the manufacturer’s instructions.