For PGC quantification, the entire embryo (E8.5-10.5) or the developing urogenital ridges (E11.5-13.5) were isolated from embryos and placed into 500 μL or 150 μL pre-warmed trypsin solution (2.5 μg.mL−1 trypsin (Gibco), 25 mM Tris, 120 mM NaCl, 25 mM KCl, 25 mM KH2PO4, 25 mM glucose, 25 mM EDTA, pH 7.6) respectively, and incubated at 37 °C for 10 min. Subsequently, 1 μL of Benzonase endonuclease (Millipore) was added and the sample gently disaggregated by pipetting and incubated for 5 min at 37 °C. The trypsin was inactivated by adding 1 mL of PBS/5% v/v fetal bovine serum and centrifuged at 1000 x g for 10 min. The sample was resuspended in 100 μL of anti-SSEA1 conjugated to Alexa Fluor 647 (catalog no. MC-480; Biolegend) diluted 1:100 in PBS/2.5% v/v fetal bovine serum and incubated for 10 min at room temperature. Samples were diluted by adding 300 μL of PBS/2.5% v/v fetal bovine serum and passed through a 70 μm filter. For quantification, 300 μL of the samples were immediately run on an ECLIPSE analyzer (Sony Biotechnology) and the data analyzed using FlowJo v10. For sorting of cells, samples were immediately run on a Synergy cell sorter (Sony Biotechnology), the cells sorted into 10 μL of PBS, centrifuged at 3500 x g for 5 min and stored at −80 °C until further analysis.
Eclipse analyzer
Manufactured by Sony
The ECLIPSE analyzer is a laboratory equipment product designed for advanced analytical tasks. It provides precise measurement and analysis capabilities without interpretation or extrapolation of the data.
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5 protocols using eclipse analyzer
1
Quantification and Sorting of PGCs
2
Isolation and Characterization of Urogenital Progenitor Cells
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Urogenital ridges of E12.5 embryos carrying the GOF18-GFP reporter were isolated and placed into 150 μl of trypsin solution (2.5 μg/ml trypsin (Gibco), 25 mM Tris, 120 mM NaCl, 25 mM KCl, 25 mM KH2PO4, 25 mM glucose, 25 mM EDTA, pH 7.6) pre-warmed to 37 °C and incubated for 10 min at 37 °C. Subsequently, 1 μl of Benzonase endonuclease (catalog no. 70664, Merk Millipore) was added per sample and samples were disaggregated by gentle pipetting and incubated for a further 5 min at 37 °C. The trypsin was inactivated by adding 1 ml of PBS + 5% fetal bovine serum (FBS). Following 10 min of centrifugation at 3300 r.p.m., the cell pellet was resuspended in 100 μl of Alexa Fluor 647-conjugated anti-human/mouse SSEA1 antibody (catalog no. MC-480; BioLegend) diluted 1:100 in PBS + 2.5% FBS and incubated at room temperature for 10 min. Then, 300 μl of PBS + 2.5% FBS were added to the cell suspension and samples were immediately run on an ECLIPSE analyzer (Sony Biotechnology) and the data analysed using FlowJo v.10.1r5 (FlowJo LLC).
3
Quantification of Primordial Germ Cells
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For PGC quantification, urogenital ridges of developing embryos (E11.5-13.5) or embryos (E9.5-10.5) carrying either the GOF18-GFP or Stella-GFP reporter were isolated and placed into 150 μl (or 500 μl in the case of E9.5-10.5 embryos) of trypsin solution (2.5 μg.ml-1 trypsin (Gibco), 25 mM Tris, 120 mM NaCl, 25 mM KCl, 25 mM KH2PO4, 25 mM Glucose, 25 mM EDTA, pH 7.6) pre-warmed to 37 °C and incubated for 10 minutes at 37 °C. Subsequently 1 μl of Benzonase (EMD Millipore) was added and the sample disaggregated by gentle pipetting and incubated for a further 5 minutes at 37 °C. The trypsin was inactivated by the addition of 1 ml of PBS, 5% v/v fetal calf serum (FCS). Following 10 minutes of centrifugation at 3,300 r.p.m., the cell pellet was resuspended in 100 μl of Alexa Fluor 647-conjugated anti-human/mouse CD15 (SSEA1) (BioLegend, MC-480) diluted 1:100 in PBS, 2.5% v/v FCS and incubated at room temperature for 10 minutes. 300 μl of PBS, 2.5% v/v FCS was added to the cell suspension and the samples immediately run on an Eclipse analyzer (Sony Biotechnology Inc.) and the data analyzed using FlowJo v10.1r5.
4
SSEA-1 Expression Analysis in Embryonic Urogenital Ridges
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Quantification was performed as described previously62 (link). Urogenital ridges of E12.5 embryos were isolated and placed into 150 μl of trypsin solution (2.5 μg ml−1 trypsin (Gibco), 25 mM Tris, 120 mM NaCl, 25 mM KCl, 25 mM KH2PO4, 25 mM glucose, 25 mM EDTA, pH 7.6) and incubated for 10 min at 37 °C. Next, 1 μl of Benzonase (Millipore) was added, followed by disaggregation of the sample by pipetting and incubation for for 5 min at 37 °C. trypsin was inactivated by adding 1 ml of PBS/5% v/v FBS. Following 10 min of centrifugation at 1,000g, the cell pellet was resuspended in 100 μl of Alexa Fluor 647-conjugated anti-human/mouse SSEA-1 antibody (catalog no. MC-480; BioLegend) diluted 1:100 in staining buffer (PBS/2.5% v/v FBS) and incubated at room temperature for 10 min; 300 μl of staining buffer were added to the cell suspension and samples immediately run on an ECLIPSE analyzer (Sony Biotechnology) and data analyzed using FlowJo v.10.1r5 (FlowJo LLC).
5
Quantification of Primordial Germ Cells
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