Fig 2: Deming regression and Bland-Altman plots of the absolute counts of T-, B-, and NK lymphocytes identified with the BD Multitest 6-Color TBNK reagent.This is the Fig 2 legend. The T- cells (CD3+, CD3+CD4+ and CD3+CD8+) shown as A, B, and C; the NK lymphocytes in D, and the B-lymphocytes are depicted in E stained with BD Multitest 6-Color TBNK reagent. The Deming regression are the upper plots with identity line (grey) and the Weighted Deming regression line (red) of each subset. The x-axis depicts BD FACSCanto II cell counts and the y-axis shows the BD FACSLyric counts. The Bland-Altman graphs are the lower plots of each subset, with the bias line (blue solid line) and the upper and lower limits of agreement (blue dotted lines). For these plots, the x-axis corresponds to the average of the lymphocyte marker, and the y-axis represents the estimated percent difference of the absolute counts between systems.

Fig 3: BD FACSLyric clinical evaluation flowchart.T-, B-, and NK cells were stained with BD Multitest reagents for testing of the 4 protocols depicted from the left to the right side: 1. Method comparison; 2. Inter-laboratory variability; 3. Determination of the reference intervals and 4. Evaluation of the specimen and stained sample storage. The summary for each protocol includes the number of specimens enrolled and cohort of origin, followed by the instrument(s) used for acquisition of the stained sample.

Fig 4: Characterization of extracellular vesicles (EVs) and validation of LNP-mRNA Incorporation into EVs. A) Flow cytometry analysis showing the percentage of purified CD63+ EVs containing Cy5-eGFP mRNA isolated from LNP-treated cells, at different time points (n = 2). One representative measurement is shown from each time point. LNP-Cy5-eGFP mRNA + beads were used instead of EVs+beads, to evaluate whether a contamination had occurred. For the negative control, only beads (without EVs or LNPs) were used. B) Detection of LNP-VEGF-A mRNA in cells and their secreted EVs, after washing out the LNPs (n = 6 for LNP-treated and UNT (untreated) cells, n = 5 for treated EVs and n = 8 for UNT EVs). Statistically significant differences were evaluated by the Mann-Whitney U-test (*p < 0.05; **p < 0.01). C) Detection of VEGF-A protein in cell lysates and supernatants, after washing out LNPs. The Kruskal-Wallis test followed by Dunn's multiple comparison test was used to compare washed versus unwashed supernatants and washed versus unwashed cells (ns = no significant differences). D) The amounts of total EV-RNA and total EV-proteins between washed and unwashed cells from LNP-treated or untreated groups (n = 6). Statistically significant differences were evaluated by the Mann-Whitney U-test (ns = no significant differences). E) Detection of CD63, and CD9-positive HTB-, HUVEC-, and CPC-EVs. Pre-enriched EVs were captured by anti-CD63-antibody-conjugated beads, and then these CD63+ EVs were captured/isolated with PE-CD9 antibody and acquired on a BD FACSLyric system (BD Biosciences). Results show that EVs that were CD63-positive were also CD9-positive. Data were analyzed using FlowJo software (TreeStar Inc.). As a negative control, CD63-antibody-conjugated beads alone (without EVs) were incubated with an equivalent volume of PBS. The experiment was performed in biological duplicates (n = 2). One representative measurement from each EV type is shown.