Cd14 pe antibody

PBMC were separated from whole blood by Pancoll (PAN-Biotech, Cat# P04-60500) density gradient centrifugation. Sorting was performed using autoMACS Pro System (Miltenyi Biotech) according to manufacturer's instructions. PBMC were stained with CD14-PE antibody (Immunotools, Cat# 21620144, clone 18D11) followed by incubation with anti-PE microbeads (Miltenyi Cat# 130-048-801). The depleted fraction was collected and resuspended in R10 medium – i.e., RPMI 1640 (Sigma-Aldrich) containing 10% heat inactivated FBS (Sigma-Aldrich), 100 U/ml penicillin, 100 U/ml streptomycin and 2 mM L-glutamine (Life Technologies), 1 mM sodium pyruvate, 100 U/ml non-essential amino-acids, 50 μM 2-mercaptoethanol (Invitrogen). 5 × 10⁵ cells/well were seeded in 96-well plates and stimulated with 20 µg/ml cGAMP or 1 µg/ml R848 (Invivogen France). For pDC depletion, PBMCs were stained with PE conjugated anti-BDCA4 (BioLegend Cat# 354504, RRID:AB_11219194) and anti-CD123 (BD Biosciences Cat# 555644, RRID:AB_396001) antibodies, and then sorted using anti-PE microbeads (Miltenyi) as above. Mock-depleted or pDC-depleted PBMCs were stimulated with R848 or ODN M362. Cell depletion was checked by flow cytometry on LSRII cytometer (BD Biosciences, RRID:SCR_002159). Supernatants were collected at 24 h and tested for IFNα by ELISA as described above.