Motorized stage

Endothelial to hematopoietic transition (EHT) was monitored by time-lapse microscopy using fluorescent optics. H1ESCs line expressing VE-Cadherin-Td tomato and CD43-GFP dual reporter were transduced with GATA2+ETV2 lentiviral constructs and cultured for 4 days until endothelial cluster were formed [13 (link)]. Time-lapse confocal imaging was performed over two days to capture blood formation. Time lapse movies were recorded using Nikon Eclipse Ti-E configured with an A1R confocal system, motorized stage (Nikon Instruments Inc. Melville, NY), and Tokai-Hit Stage Top Incubator (Tokai Hit CO., Ltd., Shizuoka-ken, Japan) at 37 °C and 5% CO₂. Images were acquired using Nikon Elements (NIS – element C) imaging software for every 5 min with CFI Plan Fluor DLL 10× NA 0.5 WD 2.1MM objective (Nikon Instruments Inc. Melville, NY). The time-lapse serial images were converted to Quick-time movies (.mov) and analyzed using ImageJ software (NIMH, Bethesda, MD).

Overnight cultures of the above strains were diluted to OD₆₀₀ of 0.05 in 3 mL of M9 medium supplemented with 0.2% casamino acids, 0.2% maltose, and 25 μM IPTG. Cells were grown at 30°C until the OD₆₀₀ reached 0.2, at which point the cells were spotted onto filter discs placed on LB agar medium supplemented with 100 μM IPTG, with or without 10 mM DTT. After 6 hours growth at 30°C, cells were suspended in liquid LB medium, fixed, and imaged using phase contrast microscopy. Scale bar, 5 μm. Where indicated, cells were fixed in 2.6% formaldehyde with 0.04% glutaraldehyde at room temperature for 1 h, followed by storage at 4°C for 24 hours. Prior to imaging, cells were immobilized on 2% agarose pads and covered with #1.5 coverslips.
Imaging was performed on a Nikon Ti inverted microscope equipped with a 100x Plan Apo 1.4 NA phase contrast objective, Andor Zyla 4.2 sCMOS camera, and Nikon motorized stage. Acquisition software was NIS Elements 4.30. The purchase of this microscope was funded in part by grant S10 RR027344–01. Microscopy was performed with the support of Microscopy Resources on the North Quad (MicRoN) at Harvard Medical School. The ImageJ plugin MicrobeJ⁴¹ was used segment cells and measure cell dimensions. Statistical significance was determined using a two-way ANOVA followed by Tukey’s Multiple Comparisons Test.