Supplementary MaterialsS1 Fig: X-VIVO serum free media best supports NK cell growth for cellular transfections. with miR-146a-5p sense or antisense miRNA compared to non-transfected control cells. A-C) Cellular viability, purity, and efficiency were determined by flow cytometry. D) MiRNA delivery was assessed by RTqPCR. Baseline reflects expression level of mi-146a-5p in cells transfected with negative control miRNA. Fold changes compared to negative were calculated using two reference miRNAs and the Pflaff Method. Data represents individual measurements and bars represent mean standard deviation, n = 1C3. RTqPCR results were assessed by one-way ratio paired tests.(DOCX) pone.0231664.s002.docx (422K) GUID:?F115641B-8C4B-49FE-A311-BBE2F86E5596 S1 Table: Qiagen miRCURY LNA sense and antisense miRNA sequences. (DOCX) pone.0231664.s003.docx (68K) GUID:?1C9E6870-C523-484B-AE54-17E1133BAE06 S2 Table: Primer sequences, efficiencies, and annealing temperatures for miRNA. (DOCX) pone.0231664.s004.docx (64K) GUID:?211284C5-B9ED-4CB5-9411-13E62CE9F988 S3 Table: Primer sequences, efficiencies, and annealing temperatures for mRNA. (DOCX) pone.0231664.s005.docx (21K) GUID:?85C42140-174E-4F75-BC71-5ED156A5C1F1 S4 Table: Flow cytometry antibodies, dyes and labels. (DOCX) pone.0231664.s006.docx (129K) GUID:?F0E2E72A-F326-467E-8528-284B97ED626B S5 Table: Non-exhaustive MiRBase sequence blast. (DOCX) pone.0231664.s007.docx (74K) GUID:?13B52FBC-2DE3-4783-89B9-89A29A821F41 Attachment: Submitted filename: and for 3 minutes to promote cell-cell contact. K562 co-cultures were incubated for 5 hours and autologous PBMC co-cultures were incubated for 2 hours with or without 5 g/mL RTX. Both co-cultures were maintained in X-VIVO 10 media with anti-LAMP1 (CD107a) antibody. To assess functional results (cytotoxicity and degranulation) co-cultures were stained for flow cytometry analysis. Statistical analysis All statistical analyses were conducted on either normalized RTqPCR relative gene expression or flow cytometry geometric means as appropriate. Samples were tested for normality with the Shapiro-Wilk normality test, and passed normality if = 0.05. If the data passed normality, analysis of variance (ANOVA) (S)-(-)-Perillyl alcohol and parametric matched ratio paired tests were completed. If the data did not pass normality, paired non-parametric Wilcoxon tests were performed. Data for all statistical tests was deemed significant if p 0.05. Results Establishment of serum-free growth conditions for primary human NK cells MiRNAs are extremely conserved, often having exact or (S)-(-)-Perillyl alcohol highly homologous sequences across mammalian species. We compared the sequences for miR-155-5p and miR-146a-5p between humans, cows, horses and mice: species whose serum is most often used in the culture of human NK cells. As expected, there is extensive inter-species conservation for these miRNA (S5 Table). To avoid introduction of extraneous miRNAs through culture and/or transfection, we developed serum-free culture conditions for primary human NK cells. NK-92 and primary human NK cells were cultured for up to four days, and cellular viability was assessed by trypan blue exclusion and flow (S)-(-)-Perillyl alcohol cytometry (S1 Fig). NK-92 cells grown in X-VIVO and RPMI maintained a viability of 95% but cells grown in ATCC recommended media exhibited a decreased viability of 85% after four days. Surprisingly, primary NK cells grown in X-VIVO media maintained a higher viability (922%) than those cultured in ATCC media (877%) after four days of culture. Cellular viabilities did not significantly differ between the ATCC recommended media for the NK-92 cell collection or primary human being NK cells and all subsequent experimentation was consequently carried out using serum free X-VIVO 10 press. TransIT-TKO outcompetes additional transfection techniques for delivering sense and antisense miRNAs to main human being NK cells To determine the best technique for main NK cell transfections, we compared the effectiveness and viability of multiple transfection techniques, including lipofectamine, nucleofection, TransIT-SiQuest, and TransIT-TKO, a reagent created for delivery of siRNA (Fig 1). We used a fluorescein (FAM)-labeled control miRNA which encodes only a scramble sequence (i.e. no specific miRNA) to compare transfection approaches. The FAM label was included in this and all transfections (control, mimic and antisense). FAM allowed us to track transfection effectiveness as the proportion of FAM+ among viable NK cells after transfection, and persistence of labeled oligonucleotides. Among the transfection methods tested, only TransIT-TKO could expose miRNA efficiently (93.4+/-2.9% transfected), and without substantial mortality among recipient cells (932.8% viable at 2.5 days post-transfection). Related transfection efficiencies were acquired for scramble oligonucleotide settings and all miRNA mimics and inhibitors used in subsequent experiments. To our knowledge, this is the most efficient transfection of NK cells reported. Open in a separate windowpane Fig 1 TransIT-TKO outcompetes additional transfection techniques.RosetteSep isolated primary human being NK cells were transfected with 25 nM FAM-labeled bad control (scramble oligonucleotide) for 24 hours using nucleofection (red circle), lipofectamine (blue square), TransIT-SiQuest (black gemstones), or TransIT-TKO (green triangles). (S)-(-)-Perillyl alcohol A-B) Cellular purity, viability, and transfection effectiveness was assessed by circulation cytometry. C-E) Assessment of transfection efficiencies Rabbit Polyclonal to PPIF and viabilities between transfection techniques. Plots represent individual transfection attempts, bars represent mean standard deviation. Data was assessed by one-way ANOVA (C) and unpaired checks (D-E). We were surprised from the high and consistent miRNA NK cell transfection efficiencies without compromise to cellular viabilities achieved by TransIT-TKO. To determine whether this reagent was universally effective for delivery of miRNA, we.