Human
blood was obtained with informed consent from healthy human donors.
PBMCs were isolated by Ficoll-Paque (GE Healthcare) density gradient
centrifugation. Human B cells were isolated by positive selection
with anti-CD19 microbeads (Miltenyi Biotec). PDCs were isolated by
positive selection with anti-BDCA-4 microbeads (Miltenyi Biotec) and
were added back to untouched PBMC resulting in final PDC percentages
of 0.5–2.4. All cells were resuspended in RPMI-1640 (BioWhittaker)
supplemented with 10% heat-inactivated FBS (Gemini) plus 50 U/mL penicillin,
50 mg/mL streptomycin, 2 mMl -glutamine, 10 mM HEPES, and
1 mM sodium pyruvate (BioWhittaker). For stimulation, B cells were
cultured at 0.75 × 106/mL in 96-well round-bottomed
plates in duplicate with CpG-ODN at a concentration range of 5.5–0.0054
μM for 90–93 h. PDC-enriched PBMC were cultured at 2.5
× 106/mL in 96-well flat bottomed plates in triplicate
with CpG-ODN at a concentration range of 2.5–0.0049 μM
for 21–24 h.
blood was obtained with informed consent from healthy human donors.
PBMCs were isolated by Ficoll-Paque (GE Healthcare) density gradient
centrifugation. Human B cells were isolated by positive selection
with anti-CD19 microbeads (Miltenyi Biotec). PDCs were isolated by
positive selection with anti-BDCA-4 microbeads (Miltenyi Biotec) and
were added back to untouched PBMC resulting in final PDC percentages
of 0.5–2.4. All cells were resuspended in RPMI-1640 (BioWhittaker)
supplemented with 10% heat-inactivated FBS (Gemini) plus 50 U/mL penicillin,
50 mg/mL streptomycin, 2 mM
1 mM sodium pyruvate (BioWhittaker). For stimulation, B cells were
cultured at 0.75 × 106/mL in 96-well round-bottomed
plates in duplicate with CpG-ODN at a concentration range of 5.5–0.0054
μM for 90–93 h. PDC-enriched PBMC were cultured at 2.5
× 106/mL in 96-well flat bottomed plates in triplicate
with CpG-ODN at a concentration range of 2.5–0.0049 μM
for 21–24 h.