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. 2021 Jul 13;12:674334. doi: 10.3389/fimmu.2021.674334

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Copyright © 2021 Angelidou, Diray-Arce, Conti, Netea, Blok, Liu, Sanchez-Schmitz, Ozonoff, van Haren and Levy

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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A human in vitro platform to assess age-dependent BCG training. Peripheral venous blood was drawn from healthy adult volunteers and cord blood was collected from healthy term (≥ 37 weeks gestation) elective cesarean deliveries. After PBMC and CBMC isolation, pure CD33+ Mos were isolated by immunomagnetic separation and plated in two separate 96-well plates. After 1h of resting, Mos were stimulated with control RPMI medium or with BCG. Supernatants were harvested from the first plate at 24h post-stimulation for Day 1 cytokine and lactate measurements. The second plate was further cultured after BCG was filtered out at 24h to allow time for immune training. After intermediate washing and culture medium replenishment steps, trained and untrained (control) monocytes were stimulated at Day 6 with LPS for 24h at which point supernatants were harvested for cytokine and lactate measurements. PBMC, peripheral blood mononuclear cells; CBMC, cord blood mononuclear cells; Mo, monocyte; sups, supernatants.