Table 1.

Protocol — procedure and timing.

	Protocol	Comments
Pre-incubation of lymphocytes with Antagomirs, timing 2.5 h
1	Wash lymphocytes (naïve or resting primary T or B cells) with cold PBS and centrifuge at 300 g for 8 min.
2	Resuspend lymphocytes (up to 1 ∗ 107 cells/ml) in 0.25 volumes of final culture volume of serum-free medium, e.g., ACCELL (Dharmacon) (i.e., if cell culture is carried out in 5 ml, resuspend lymphocytes in 1.25 ml serum-free medium).	Critical: serum-free medium increases the efficiency of Antagomir uptake from the medium. The cholesterol-coupled Antagomirs will attach to the cell surface and are subsequently incorporated.
3	Dilute Antagomirs at the desired and titrated concentration.	Critical: even at only moderate miRNA knockdown efficiency there may already be visible phenotypical changes and impact on potential targets. Too high concentrations of Antagomirs may lead to off-target effects. Therefore, a titration of the Antagomirs and determination of suitable analysis time points in different culture settings is highly recommended prior to experimental procedures.
4	Gently mix samples and incubate for 2 h at 5% CO2, 95% humidity and 37 °C. Meanwhile prepare stimulating and differentiating medium for further cell culture.
Stimulation and culture of Antagomir-treated lymphocytes in vitro
5	Add 0.75 volumes of serum-containing culture medium supplemented with all necessary cytokines and stimuli to culture wells/plates (e.g., in this study, repeatedly activated Th1 cells were restimulated with plate-bound αCD3/αCD28 and recombinant IL-12 and anti-IL-4 antibody).	Antagomir uptake does not depend on reactivation of lymphocytes. Resting lymphocytes treated with Antagomirs can also be monitored (e.g., Fig. 1d) and analyzed.
6	Add 0.25 volumes of Antagomir-treated cells to the medium and incubate at 5% CO2, 95% humidity and 37 °C for the desired time.	Critical: Determine the most suitable analysis time point prior to experimental procedures, e.g., by expression kinetics of miRNA, target genes and usage of reporter gene assays.
Control knockdown efficiency by quantitative real-time PCR analysis
7	At the end of the experimental procedure, perform the desired analysis and take an aliquot of the cells to determine knockdown efficiency of Antagomirs (and their potential impact on targets) by qRT-PCR analysis or Northern Blots (and/or protein detection).