Flow cytometry data will be analyzed using the software package FlowJo and the achieved transduction efficiency (%GFP+ cells) will be calculated for each infected cell population. Protocol 1, the results of which will be compared to Physique S11C. Next, the viral transduction of GSCs and U87MG Rabbit Polyclonal to ANKK1 cells with expression constructs for Tie2-and PGK-expression in various cell lines using qPCR This protocol evaluates the expression of the endothelial marker in three cell lines using semi-quantitative PCR: patient-derived glioblastoma neurospheres (GSC83), human glioblastoma cell line U87MG, and normal human dermal microvascular endothelial cells (HMVEC-d). The expression of will be normalized against the endogenous expression of 18S rRNA. Expression of is usually expected to be very low in GSC83 and U87MG cells, and robust in the endothelial cell line HMVEC-d, as depicted in Physique S11C. This protocol serves as a quality control step to ensure the lack of expression in the glioblastoma cell lines used later in the study. Sampling This experiment will be performed three times (biological replicates) with each run using two technical replicates, for a final power of at least 80%. A. Test conditions: i. qRT-PCR of (and 18S rRNA) from GSC83 glioblastoma neurospheres. ii. qRT-PCR of (and 18S rRNA) from U87MG cells. iii. qRT-PCR of (and 18S rRNA) from HMVEC cells. Materials and reagents expression levels across cell types using a StepOnePlus Real-Time PCR System. Use 18S rRNA as an endogenous control. Perform duplicate technical replicates for each biological replicate (3 biological 2 technical 2 genes = 12 wells per VU0652835 cell line). A. Use 1 l of undiluted cDNA mixture for each reaction. B. Use TaqMan probes for and 18S rRNA (see reagent table). C. Use an initial denaturation at 95C for 10 min, following by 40 cycles of 95C for 15 s; 60C for 1 min. Analyze and compute CT values. Deliverables Data to be collected: A. Purity (A260/280 and A260/230 ratios) and concentration of isolated total RNA from cells. B. Raw qRT-PCR values, as well as analyzed CT values and bar graph of mRNA normalized to control mRNA levels for each condition (compare to Figure S11C). Confirmatory analysis plan This replication attempt will perform the statistical analyses listed below, compute the effect sizes, compare them against the reported effect size in the original paper and use a meta-analytic approach to combine the original and replication effects, which will be presented as a Forest plot. Statistical analysis of the replication data: A. One-way ANOVA to analyze the means of GSC83, U87MG, and HMVEC. i. We will then perform a Fisher’s LSD test to perform multiple pairwise comparisons: a. GSC83 compared to HMVEC. b. U87MG compared to HMVEC. c. GSC83 compared to U87MG (sensitivity). Known differences from the original study In the original study, multiple human glioblastoma neurospheres were screened for expression. The human glioblastoma cell lines U251 and T98G were also analyzed, as well as the human endothelial cell line HUVEC. This replication study will be using a single established glioblastoma neurosphere cell line (GSC83) provided by the authors. The authors will also provide their U87MG and HMVEC cell lines. All known differences in reagents and supplies are listed in the Materials and reagents section above, with the originally used item listed in the Comments section. All differences have the same capabilities as the original and are not expected to alter the experimental design. Provisions for quality control The cell lines used in this experiment will undergo STR profiling to confirm their identity and will be sent for mycoplasma testing to ensure there is no contamination. The sample purity (A260/280 and A260/230 ratios) of the isolated RNA from each sample will be reported. All data obtained from the experimentraw data, VU0652835 data analysis, control data, and quality control datawill be made publicly available, either in the published manuscript or as an open access dataset available on the Open VU0652835 Science Framework project page for this study (https://osf.io/mpyvx/). Protocol 2: Lentiviral contamination of glioblastoma cells and stable.