For knockdown experiments, cells from E10 dorsal cortices were counted and resuspended with buffer R (Neon™ transfer system, Invitrogen) in the concentration of 8 × 106 cells/mL. Resuspended cells (100 µL) were mixed with 160 µM control or Fbl siRNA. Electroporation was performed with Neon (Neon™ transfer system, Invitrogen) at conditions of 1600 voltage, 20 width and 1 pulse. These transfected cells were mixed with 2 ml culture medium (20 ng/ml human basic FGF (Peprotech), 1XB27 RA- (Gibco), in Dulbecco’s Modified Eagle Medium (DMEM) F12 + GlutaMax (Gibco) and distributed into 4-well or 24-well plates (500 µL/well) and cultured at 37 °C. For inhibitors treatment, 2 × 105 cells/well were culture in the DMSO, 0.5 µM GSK-J4 (Sigma, SML0701), 2.5 µM GSK-343 (Sigma, SML0766), or 0.5 µM GSK-J4 and 2.5 µM GSK-343 for 3 days.
1xb27 ra
Manufactured by Thermo Fisher Scientific
The 1XB27 RA- is a laboratory equipment product manufactured by Thermo Fisher Scientific. It is a device designed for specific laboratory applications. The core function of this product is to perform tasks related to the intended use, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Human fgf basic, by Thermo Fisher Scientific (2 mentions)
Dmem f 12 glutamax, by Thermo Fisher Scientific (2 mentions)
Gsk j4, by Merck Group (1 mentions)
Neon transfer system, by Thermo Fisher Scientific (1 mentions)
Gsk343, by Merck Group (1 mentions)
Countess 2, by Thermo Fisher Scientific (1 mentions)
35 m filter, by BD (1 mentions)
Genomics chromium, by 10x Genomics (1 mentions)
Countess, by Thermo Fisher Scientific (1 mentions)
Sh800, by Sony (1 mentions)
2 protocols using 1xb27 ra
1
Fbl Knockdown in Cortical Cells
2
Isolation and Sorting of Hes1+ Neural Stem Cells
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To isolated Hes1 positive neural stem cells (NSCs) at E14, dorsal cortices were dissected from Hes1-d2-EGFP Tg/+ mice. Cortices were dissociated with 0.05% trypsin with Hanks'Balanced Salt Solution (HBSS) (-) at 37 °C for 10 min. After centrifugation at 1000 g for 5 min, cells were resuspended with 0.375% BSA/HBSS(-) by gentle pipetting 15 to 20 times. Resuspended cells were filtered with 35 µm filter (Falcon) and sorted into sorting buffer (20 ng/ml human basic FGF (Peprotech), 1XB27 RA-(Gibco), in Dulbecco's Modified Eagle Medium (DMEM) F12+GlutaMax (Gibco)) by a cell sorter (SH800, SONY) equipped with 130 µm sorting chips (SONY, LE-C3113). After sorting, cell number was counted by Countess or Countess II (Invitrogen). For cells from wild type and Fbl mutant mice, cells were collected similarly except that sorting was not performed.
Collected cells were immediately load into the 10X-Genomics Chromium (10X Genomics, Pleasanton, CA). Libraries for single cell cDNA were prepared using Chromium 3′ v2 platform as the manufacturer′s protocol.
Collected cells were immediately load into the 10X-Genomics Chromium (10X Genomics, Pleasanton, CA). Libraries for single cell cDNA were prepared using Chromium 3′ v2 platform as the manufacturer′s protocol.
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