160 | Chapter 6 diluted 1:5 with PCR-grade water. The PCR master mix contained 5 μl of Sso Fast EvaGreen supermix (Bio-Rad, Hercules, CA) and 0.5 μl of 10 μM reverse and forward primers (Biolegio, Nijmegen, the Netherlands). The final volume was adjusted to 9 μl using PCR-grade water, to which 1 μl of diluted cDNA was added. The following PCR conditions were used: initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing at 65°C for 5 s, and extension at 72°C for 10 s. The fluorescent product was measured by a single acquisition mode at 72°C after each cycle. For distinguishing specific from nonspecific products and primer dimers, a melting curve was obtained after amplification by holding the temperature at 65°C for 15 s followed by a gradual increase in temperature to 95°C at a rate of 2.5°C s−1, with the signal acquisition mode set to continuous. For each cDNA preparation, PCRs were performed at least in duplicate, and for each condition, at least three independent experiments were performed. Expression of target gene mRNAs was normalized to the housekeeping genes large ribosomal protein PO (RPLPO) for human samples and 18S rRNA (18S rRNA) for mouse samples. Expression of miR-34a was assessed by Taqman MicroRNA assay and normalized to snoRNA429 (Applied; now ThermoFisher) as per manufacturer’s protocol. The following primers were used: mouse Rag1-forward (5’-AGCTATCACTGGGAGGCAGA-3’), mouse Rag1-reverse (5’-GAGGAGGCAAGTCCAGACAG-3’), mouse Rag2-forward (5’-CTTCCCCAAGTGCTGACAAT-3’), mouse Rag2-reverse (5’-AGTGAGAAGCCTGGCTGAAT-3’), mouse Foxp1-forward (5’-AGTCATGATGCAAGAATCTGGGT-3’), mouse Foxp1-reverse (5’- TCTCCGTTGGACCGAGTGTCACG-3’) mouse 18S rRNA-forward (5’-TGGTGGAGGGATTTGTCTGG-3’), and mouse 18S rRNA-reverse (5’-TCAATCTCGGGTGGCTGAAC-3’). The data were analyzed in GraphPad, on figures expressed as mean±SD, ANOVA statistical analysis was used for multiple comparisons with the alpha threshold of 0.05. Where data are compared as fold induction/reduction against a control group, a non-parametric Kruskal-Wallis test was used with the alpha threshold set to 0.1 due to a low number of repeats. FACS Flow-cytometry stainings and analyses were performed according to the protocol as described earlier8. Briefly, 1x106 cells were washed 2x with 2mL of cold FACS buffer (1x PBS + 0. 2% NaN3 + 1% FCS) and spun at 1100 rpm for 5 minutes. After the addition of the antibodies, the cells were incubated on ice in the dark for 30min and then washed again with the cold FACS buffer. Next, the cells were resuspended in 1 mL 1x PBS + 1% paraformaldehyde and incubated for 5 minutes at room temperature. Finally, the cells were spun at 1100rpm and re-suspended again in 400 mL of FACS buffer. The doublets were excluded based on forward and side scatter height versus width profiles. Flow cytometry was performed on a four-laser, thirteen-detector LSR Fortessa (BD Biosciences). Flow cytometry data were analyzed using FlowJo (Tree Star Inc., Ashland). The following antibodies were used to
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