Journal: STAR protocols
Article Title: Protocol for extracting live blastoderm cells from embryos of annual killifish.
doi: 10.1016/j.xpro.2023.102344
Figure Lengend Snippet: Figure 4. Yield of A. nigripinnis blastoderm cells (deep embryonic and EVL cells) extracted at early post epiboly stages (A) Quantification of cell viability after extraction using the Neubauer chamber. Live cells appear green as they retain Calcein-AM fluorophore. Large cells are EVL cells and small are deep cells. (B) Exemplary images of a quadrant of the Neubauer chamber (left) and enlarged view (right) illustrate how to recognize EVL and deep cells and how to distinguish between live cells (those that retain Calcein-AM – green arrows) and dead cells (without fluorescence – red arrow). Images were acquired using a Nikon Ti2 eclipse inverted microscope with a 103 air objective with 0.30 NA. Scale bar: 100 mm. (C) Comparison of cell size allows distinguishing EVL (diameter above 10 mm) from deep cells (about 5 mm in diameter). Live cells also typically display cellular activities such as presence of cellular extrusion. Images were acquired using a Nikon Ti2 eclipse inverted microscope with a 403 air objective with 0.95 NA. Scale bar: 20 mm.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Chemicals, peptides, and recombinant proteins SYLGARD 184 Silicone Elastomer Kit (PDMS) Dow Corning Cat#101697 Pluronic F-127 Sigma-Aldrich Cat#P2443 (Continued on next page) 2 STAR Protocols 4, 102344, September 15, 2023 Continued REAGENT or RESOURCE SOURCE IDENTIFIER NaCl Winkler Cat#SO-1455 KCl Winkler Cat#302407 CaCl2/2H2O Winkler Cat#CA-0520 NaHCO3 Sigma-Aldrich Cat#S5761-1KG MgCl2/6H2O Winkler Cat#MA-0960 Tris base Sigma-Aldrich Cat#10708976001 EGTA Sigma-Aldrich Cat#E8145 Pronase Sigma-Aldrich Cat#10165921001 DMEM/F-12 powder (L-Glutamine – 15mM HEPES – without NaHCO3 and without phenol red) Sigma-Aldrich Cat#D2906 Pen/strep Gibco Cat#15140122 10% Chlorine solution Avalco Cat#4201 Calcein AM Thermo Fisher Cat#C3099 Experimental models: Organisms/strains Austrolebias nigripinnis 4,7 NCBI:txid135282 Nothobranchius furzeri 4,16 NCBI:txid105023 Other Dumont Dumoxel Style 5/45 Tweezer, Biology Tips, Antimagnetic Stainless Steel, 109 mm Spi Supplies Cat#0T054A-XD Leica S6 D Stereoscope Leica Microsystems https://www.leica-microsystems. com/products/light-microscopes/ stereo-microscopes/p/leica-s6-d/ Thermo Scientific Heraeus Pico 17 microcentrifuge Thermo Scientific Cat#75002401 Nikon Eclipse Ti2 inverted microscope Nikon instruments https://www.microscope. healthcare.nikon.com/ products/invertedmicroscopes/eclipse-ti2-series Weighing scale Belltronic XU1002B Cat#B-01-02-03-0401 Vacuum pump Rocker 300 Cat#167300 Vacuum desiccator Sp Bel-Art "space saver" polycarbonate vacuum desiccator Cat#F42025-0000 Forced air drying oven Biobase BOV-V65F Cat#AO-BOV-V65F Plasma cleaner Tergeo - Pie Scientific – UV light source Ossila UV ozone cleaner Cat#L2002A3 Stereoscope Leica S6D Cat#S6D-PS Cell culture centrifuge Thermo Scientific Heraeus Pico 17 Cat#75002414 UV Ozone Cleaner Ossila Cat#L2002A3-UK Tissue-culture treated culture dishes, 35 mm Corning Cat#CLS430165 Biopsy punch, sterile, 8 mm, 10/pk Kruuse Cat#273693 Disposable surgical blades size 10 ChannelMed Cat#CJ6011 Style #2A Flat Tipped Tweezers, Antimagnetic Stainless Steel, High Precision, 120 mm SPI-Swiss Cat#0S2AP-XD 1.0 mL volume, flat bottom 9 3 38 mm soda glass tube with push in cap Teklab Cat#GL389 Pasteur pipettes Nest Cat#318212 Pipet-Lite XLS+ manual single-channel pipette, 20–200 mL Rainin Cat#17014391 Flat tipped No. 10 brush Artel Cat#20660140 15 mL Conical centrifuge tubes Falcon Cat#14-959-53A Microtubes 38103 Eppendorf Cat#Z606340 ll OPEN ACCESS STAR Protocols 4, 102344, September 15, 2023 3 Protocol ll OPEN ACCESS Protocol
Techniques: Extraction, Inverted Microscopy, Comparison
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