FIGURE 3.

Smpd3 deletion in mixed cortical cell (MCC) cultures does not affect the level of EV production. (a) Schematic overview of the experimental set‐up for Smpd3 ^fl/fl MCC culture, TAT‐CRE mediated Smpd3 deletion and EV collection. (b) Deflox PCR on lysed MCC cells. The presence of the deletion band (402 bp) shows effective TAT‐CRE mediated ‘defloxing’. The flox band (2121 bp) is nearly absent in TAT‐CRE induced Smpd3 KO MCCs (Smpd3 KO) while still present in the Smpd3 WT MCCs (Smpd3 WT). (c) Smpd3 expression in Smpd3 KO MCCs analysed with RT‐qPCR, relative to Smpd3 WT MCCs. (d–h) ExoView quantification of CD9⁺ EVs in MCC culture medium from Smpd3 WT (n = 3) and Smpd3 KO (n = 3) MCC cultures, showing the total (d), CD81⁺ (e), CD63⁺ (f), CD9⁺ (g) and label‐free scatter⁺ (50–200 nm) (h) EVs on the CD9 capture spot. (I–M) Expression level of Smpd1 (i), Smpd2 (j), Alix (k), Tsg101 (l) and Stam1 (m) in MCCs from Smpd3 WT (n = 4) versus Smpd3 KO mice (n = 3). Results are represented relative to Smpd3 WT MCC expression values. Data are represented as means ± SEM. Statistical comparison of two groups was performed by unpaired t‐testing or a Mann–Whitney test (**p < 0.01; ns, not significant). Alix, apoptosis linked gene 2 interacting protein X; EV, extracellular vesicle; MCC, mixed cortical cell; Smpd, sphingomyelin phosphodiesterase.