46C mESCs (3×105) were seeded in Fibronectin (16.7 μL/mL, F1141-5MG, Sigma) coated plates and cultured in N2B27 medium supplemented with 20 ng/mL Activin A (C678, Novoprotein, China), 12 ng/mL bFGF (C044, Novoprotein, China) and 1% KSR (10828028, Invitrogen) to induce epiblast like stem cells (EpiLCs). After two days, 2×105 EpiLCs were inoculated into GK15 medium for suspension culture for 4 days to induce PGCLCs in the presence of the inductive cytokines BMP4 (500 ng/mL, 315-27-10, Peprotech), LIF (1000 U/ml, Millipore), SCF (100 ng/mL, AF-250-03, Peprotech) and EGF (50 ng/mL, AF-100-15, Peprotech). GK15 consists of GMEM (11710035, Gibco), 15% KSR, 1×MEM nonessential amino acids, 0.1 mM β-mercaptoethanol and 1 mM sodium pyruvate (SP0100, Solarbio, China). After dissociation, the PGCLCs were resuspended in 1×PBS and analyzed on flow cytometers (FACS Calibur, BD Biosciences).
Af 250 03
Manufactured by Thermo Fisher Scientific
The AF-250-03 is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is a compact and portable device designed for general laboratory applications. The core function of the AF-250-03 is to provide accurate and reliable measurements, but a detailed description of its intended use is not available.
Automatically generated - may contain errors
Lab products found in correlation
F1141 5mg, by Merck Group (3 mentions)
Af 100 15, by Thermo Fisher Scientific (3 mentions)
Activin a, by Novoprotein (2 mentions)
Basic fibroblast growth factor (bfgf), by Novoprotein (2 mentions)
Sp0100, by Solarbio (1 mentions)
Facscalibur, by BD (1 mentions)
N1250, by Solarbio (1 mentions)
P1400, by Solarbio (1 mentions)
3 protocols using af 250 03
1
Differentiation of mESCs to PGCLCs
2
Directed Differentiation of PGCLCs
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First, 46C ESCs (3 × 105) were seeded in plates coated with fibronectin (16.7 μl/ml, F1141-5MG, Sigma) and cultured in serum-free N2B27 medium with 20 ng/ml Activin A (C678, Novoprotein, China), 12 ng/ml bFGF (C044, Novoprotein, China) and 1% KSR (10828028, Invitrogen) to induce EpiLCs. Two days later, 2 × 105 EpiLCs were exposed to PGCLC-inductive medium containing BMP4 (500 ng/ml, 315-27-10, Peprotech), LIF (1000 U/ml, Millipore), SCF (100 ng/ml, AF-250-03, Peprotech), EGF (50 ng/ml, AF-10015, Peprotech), 15% KSR and GMEM medium for 4 days to induce PGCLCs.
3
Protocols for Generating Mouse and Human PGCLCs
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We mainly referred to two previous studies for the induction protocols (14 (link), 18 (link)). Briefly, for mouse PGCLC formation, 1 × 105 46C ESCs were seeded in fibronectin bovine plasma (16.7 μl/ml, F1141-5MG, Sigma) coated plate and were cultured in activin A (20 ng/ml, C678, Novoprotein) and basic fibroblast growth factor (12 ng/ml, C044, Novoprotein) containing medium to induce EpiLCs. The medium was changed every day. After 2 days, 3 × 105 EpiLCs were incubated in GK15 medium, consisting of Glasgow's minimal essential medium (11710035, Gibco), 15% KSR (10828028, Invitrogen), 1× penicillin/streptomycin (P1400, Solarbio), 1× MEM nonessential amino acids, 0.1 mM β-mercaptoethanol, and 1 mM sodium pyruvate (N1250, Solarbio), to generate PGCLCs in the presence of BMP4 (500 ng/ml, 315-27-10, PeproTech), LIF (1000 U/ml, Millipore), SCF (100 ng/ml, AF-250-03, PeproTech), and EGF (50 ng/ml, AF-100-15, PeproTech).
For human PGCLC induction, 1 × 105 human iPSCs were differentiated into iMeLCs with 50 ng/ml activin A, 3 mM CHIR99021, and 10 mM ROCK inhibitor (Y27632). iMeLCs (3 × 105) were then incubated in GK15 medium supplemented with BMP2 (200 ng/ml, C012, Novoprotein) or BMP4 (500 ng/ml, 120-05-5, PeproTech), LIF (1000 U/ml, Millipore), SCF (100 ng/ml, C034, Novoprotein), EGF (50 ng/ml, AF-100-15, PeproTech), and 10 mM ROCK inhibitor for 4 days.
For human PGCLC induction, 1 × 105 human iPSCs were differentiated into iMeLCs with 50 ng/ml activin A, 3 mM CHIR99021, and 10 mM ROCK inhibitor (Y27632). iMeLCs (3 × 105) were then incubated in GK15 medium supplemented with BMP2 (200 ng/ml, C012, Novoprotein) or BMP4 (500 ng/ml, 120-05-5, PeproTech), LIF (1000 U/ml, Millipore), SCF (100 ng/ml, C034, Novoprotein), EGF (50 ng/ml, AF-100-15, PeproTech), and 10 mM ROCK inhibitor for 4 days.
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