Monocyte-derived Dendritic Cell Culture

Blood packs were acquired from the National Health Service Blood and Transplant Service (NHSBTS) an HTA licenced provider. Monocytes were isolated using CD14 microbeads (Miltenyi Biotec) and the MACS system (Mitenyi Biotec). After treatment with 1500U/ml IL4 (Bio-Techne) and 400U/ml GMCSF (Bio-Techne), cells were cultured in AIM-V media (GIBCO) for 6 days. Cells were confirmed to be immature DCs using flow cytometry as described^{27 (link)}. Extract was diluted in PBS to reach stock concentration for use on cells. Heparin sulphate (Sigma) was dissolved in PBS before use. Cell viability was assayed using Annexin-V/PI (BD biosciences) staining by flow cytometry.

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Lynham S., Freile F.G., Puri N.M., O’Reilly N., Mitchell G.H., Wells T.N., Willcox M, & Beatson R. (2021). Identification of chlorophyll a-b binding protein AB96 as a novel TGFβ1 neutralizing agent. Scientific Reports, 11, 7740.

Publication 2021

CD14 microbeads IL4 GMCSF AIM-V media Heparin sulphate Annexin-V/PI

After treatment Annexin v Blood Cell viability Cells Cultured media Flow cytometry Gmcsf Heparin Microbeads Monocytes National health service Sulphate Transplant

Corresponding Organization :

Other organizations : King's College London, University of Essex, Medicines for Malaria Venture, University of Southampton

Top 5 similar protocols

Variable analysis

independent variables

IL4 concentration (1500 U/ml)
GMCSF concentration (400 U/ml)

dependent variables

Immature dendritic cell phenotype (confirmed by flow cytometry)
Cell viability (assayed using Annexin-V/PI staining)

control variables

Monocyte isolation method (CD14 microbeads and MACS system)
Cell culture media (AIM-V)
Cell culture duration (6 days)

positive controls

None specified

negative controls

None specified

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