Prior to dissociation, to focus on tissues that contribute to the embryo proper, the ectoplacental cone, parietal endoderm, and much of the extraembryonic ectoderm, were removed using fine forceps. For isolation of single cells for scRNA-seq, embryos were dissociated with 0.25% Trypsin-A, 0.02% EDTA (Biological Industries) solution for 5′ at 37°C, and resuspended in DMEM w/o phenol red (GIBCO) supplemented with 10% FBS (Biological Industries). Samples were run on a FACSAria-III flow cytometer (BD Biosciences) using the ‘index sort’ option to retain the spectral properties of each individual sorted cell.
Trypsin a
Manufactured by Sartorius
Trypsin-A is a laboratory enzyme used to dissociate adherent cells from cell culture surfaces. It is commonly used in cell biology research and cell-based assays.
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Lab products found in correlation
2 protocols using trypsin a
1
Single-cell isolation for scRNA-seq
2
Cell Uptake Capacity on Tunable Substrates
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Cells were seeded on fabricated polyacrylamide gels and commercial gels of varying stiffnesses for 12 and 96 h. Fluorescently labeled polystyrene particles were diluted 1000-fold, added to the cells, and incubated for 6 (Fig. S5) or 12 (Fig. 5 ) h at 37 °C with 5% CO2. After incubation, the cells were washed with cold PBS, detached using trypsin A (Biological Industries, Catalog No. 03–050-1A), washed again, and filtered through a 40–50 μm nylon mesh to remove cell debris. Cells were then centrifuged and suspended in a Fluorescence-activated cell sorting (FACS) buffer containing 1% Bovine Serum Albumin (BSA, VWR Chemicals, Solon, OH, USA) and 0.05% sodium azide n 1xPBS. Cell uptake capacity was quantified using ImageStreamX Mk II (Luminex, Austin, Texas, USA) and analyzed using IDEAS software. Focused cells were defined as cells with a gradient of root mean square (RMS)≥50. Single cells were identified as cells with a high aspect ratio and low area. Bead uptake was evaluated using fluorescent channel intensity histograms, where the y-axis represents the percentage of the total cell population that internalized any number of particles.
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