Peripheral Blood Mononuclear Cell Isolation for BTV Study

Peripheral blood mononuclear cells (PBMCs) were obtained from 100 ml of total blood collected in EDTA from jugular vein at day 0 prior to infection and days 3, 7 and 15 post-BTV infection. The samples were purified using Ficoll cushion (GE Healthcare) purification method as described in (36 (link)). PBMCs were frozen as described in (36 (link)). When necessary, PBMCs were cultured in PBMC culture medium (RPMI supplemented with 10% FBS (Sigma), 4 mM L-glutamine, 10 mM HEPES, 1% 100X non-essential aminoacids, 1 mM sodium pyruvate, 100U/mL penicillin/100 μg/mL streptomycin and 50nM β-mercaptoethanol (all from Invitrogen)).

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Louloudes-Lázaro A., Rojas J.M., García-García I., Rodríguez-Martín D., Morel E., Martín V, & Sevilla N. (2023). Comprehensive immune profiling reveals that Orbivirus infection activates immune checkpoints during acute T cell immunosuppression. Frontiers in Immunology, 14, 1255803.

Publication 2023

Ficoll cushion PBMC culture medium FBS streptomycin β-mercaptoethanol

Blood Cultured medium Edta Essential aminoacids Ficoll Frozen Glutamine Hepes Infection Jugular vein Mercaptoethanol Penicillin Peripheral blood mononuclear cells Pyruvate Sodium Streptomycin

Corresponding Organization :

Other organizations : Consejo Superior de Investigaciones Científicas, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Universidad Complutense de Madrid

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Variable analysis

independent variables

Time of blood sample collection (day 0 prior to infection, days 3, 7, and 15 post-BTV infection)

dependent variables

Peripheral blood mononuclear cells (PBMCs)

control variables

Volume of total blood collected (100 ml)
Blood collection site (jugular vein)
Anticoagulant (EDTA)
PBMC purification method (Ficoll cushion)
PBMC cryopreservation method
PBMC culture medium (RPMI supplemented with 10% FBS, 4 mM L-glutamine, 10 mM HEPES, 1% 100X non-essential aminoacids, 1 mM sodium pyruvate, 100U/mL penicillin/100 μg/mL streptomycin and 50nM β-mercaptoethanol)

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