Humannaive cd8 t cell isolation kit

Peripheral blood
mononuclear cells were isolated
from buffy coats obtained from HLA-A2.1⁺ healthy volunteers
after written informed consent and in agreement with institutional
guidelines using Ficoll density gradient centrifugation (Lymphoprep,
ELITechGroup). CD14⁺ monocytes and naïve CD8⁺ T cells were isolated using CD14 microbeads and the human
Naive CD8⁺ T cell isolation kit (both Miltenyi Biotec),
respectively. Monocytes were differentiated into immature monocyte-derived
DCs (moDCs) in 6 days using RPMI 1640 containing 10% FCS, 2 mM l-glutamine, 0.5% antibiotic–antimycotic enriched with
interleukin-4 (IL-4, 300 U/mL), and GM-CSF (450 U/mL) (both Miltenyi
Biotec). Isolated cells were stored in liquid nitrogen in 10% DMSO
(CryoSure) and 90% FBS.
Human moDCs were thawed from liquid
nitrogen. In vitro DC activation (24 and 48 h) in
alginate cryogels containing NY-ESO-1 PLGA NPs was studied, as described
for BMDCs. Soluble NY-ESO-1 peptide and TLR ligands were added to
the controls in the same amounts as is present in NPs. Flow cytometric
staining was performed using Zombie Violet Fixable viability dye for
30 min at 4 °C, followed by cell-surface staining with the following
antibodies for 30 min at 4 °C: CD14-FITC (Miltenyi Biotec), CD80-PerCP-eFluor710
(PharMingen), CD86-PeCy7 (PharMingen), and CD40-PE (Beckman).