glass bottom 384 well microscopy plate  (Azenta)

Journal: bioRxiv

Article Title: Efficient Generation of Functional TCRαβ + Cytotoxic T Cells from hiPSCs via Small-Molecule Modulation

doi: 10.64898/2026.03.31.715684

Figure Lengend Snippet: A: Schematic overview of the screening approach. hiPSC-derived CD34 + hemogenic endothelial cells were differentiated towards ProT cells for 14 days in a stroma-free protocol . At iT day 14 cells were harvested and replated into a 384 well plate and exposed to a small molecule library. After 7 days cells were stained with anti CD56, anti CD4, anti CD7 with 7-AAD and Calcein Violet viability dyes. Scale bar indicates 20 µm. B: Primary screening results. 5 hits were identified when a Z-score threshold of 1 was applied. C: Hit validation, bar graphs depicting mean percentage of CD4 + cells out of CD45 + population on iT day 28 in hiPSC line EZH1 mut/wt (screening line), BCH1157 and BCH1566. Error bars depict SEM of replicate wells, one way ANOVA comparison against DMSO. E: Exemplary flow cytometry plots on iT day 28 demonstrating an increased proportion of CD4 + CD8 + DP cells after GNF351 treatment in BCH1157 hiPSC-derived ProT cells. F: Mean values plotted from two independent experiments with either continuous drug treatment (solid circles) or one time drug exposure (open circles). SEM of technical replicates is indicated by blue shading. Two-way ANOVA with Dunnett’s multiple comparison test against respective untreated cells.

Article Snippet: Rows A-O of a glass bottom 384-well plate (Cellvis, #P384-1.5H-N) were coated with DLL4 (Thermo Fisher) and VCAM1 by adding 25 μl/well with a 300 μl 12-channel pipette followed by spinning in a swing-out bucket rotor for 5 min at 300g and incubation in a cell culture incubator for 1h at 37 °C.

Techniques: Derivative Assay, Staining, Biomarker Discovery, Comparison, Flow Cytometry