SKP isolation, culture and sciatic nerve implantation protocols were performed as previously described (50 (link)). Briefly, dorsal skin was cut to yield about 1 square inch of tissue and was dissected into small pieces. To isolate SKP cells, the suspension of tissue pieces was centrifuged (30 sec, 1200 rpm), supernatant was discarded and resuspended in a solution of PBS (Sigma, D8662) (8 mL) and collagenase I (2 mL) by inversion and let it moderately shake (37°C, 40 min). Next, tissue pieces were mechanically disrupted by up and down pipetting (10 times), were centrifuged (10 min, 2000 rpm, 4°C), supernatant was discarded and was resuspended in ice cold HBSS (10 mL). The procedure was repeated twice starting back at the mechanical pipetting disruption step. Finally, the resuspension was filtered on a 70 um sterile cell strainer and viable cells were counted (trypan blue). To enrich for SKPs, 1 × 106 cells per 10 cm dish were seeded on ultra-low culture dish (Corning, 3262). After 3–4 days, half of the supernatant (5 mL) was discarded and fresh SKP complete media (5 mL) was added.
Ultra low culture dish
Manufactured by Corning
The Ultra-low culture dish is a specialized laboratory equipment designed for cell culture applications. It features a unique surface treatment that minimizes cell attachment, promoting the growth of cells in suspension.
Automatically generated - may contain errors
Lab products found in correlation
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (1 mentions)
Neurobasal medium, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Dmem medium, by Merck Group (1 mentions)
Pdgfα, by Thermo Fisher Scientific (1 mentions)
Fv1000, by Olympus (1 mentions)
4 protocols using ultra low culture dish
1
SKP Isolation, Culture, and Implantation
2
Primary Culture of Oligodendrocyte Precursor Cells
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Primary culture of OPCs was prepared according to literature 26 (link). Postnatal rats (P1) were decapitated after euthanasia procedure with CO2. Brain cortex isolated from postnatal rat was cut into 1mm3 pieces in cold Hank's Balanced Salt Solution (HBSS, Thermo Scientific), and digested in DNase I and trypsin for 15 min at 37 °C. Mixed primary cells were cultured with DMEM20S (DMEM medium supplemented with 4 mM L-glutamine, 1 mM sodium pyruvate, 10% C-FBS, 50 U/ml penicillin and 50 µg/ml streptomycin, from Sigma-Aldrich and Thermo Scientific) in poly-D-lysine-coated flasks (Sigma-Aldrich) for 10d. Microglial cells were removed by shaking on a horizontal orbital shaker at 200 rpm 37 °C overnight. On the second day, supernatant was collected and cultured with ultra-low culture dish (Corning) to further remove contaminated microglia and fibroblasts. Finally, OPCs were ready for in vitro experiments after 5-6 d in the proliferation medium (Neurobasal medium supplemented with 1:50 B27 Supplement, 1:100 GlutaMax, 10 ng/ml bFGF and 10 ng/ml PDGFα, from Thermo Scientific and PeproTech), or for differentiation in differentiation medium (proliferation medium without bFGF and PDGFα).
3
iPSC Differentiation into Lineages
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iPSCs were treated with 0.05%Trypsin/EDTA and then cultured in ES medium without LIF in Ultra Low Culture Dish (Corning) for 5 days. Then the EB were transferred to gelatin-coated 24-well plate and cultured for 14 days. Immunostaining were performed with antibodies for specific lineage markers. Images were captured under a confocal microscope (Olympus FV1000) at 60× magnification.
4
Colorectal Cancer Sphere-Formation Assay
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HT29 human colorectal cancer cells were plated at a density of 2 × 106 cells per Ultra‐Low Culture Dish (Corning, One Riverfront Plaza, Corning, NY) for tumor sphere‐formation assays. Cells were grown in RPMI‐1640 medium supplemented with 1 × B27 supplement (Invitrogen), 20 ng/mL epidermal growth factor (EGF; Peprotech), 10 ng/mL basic fibroblast growth factor (bFGF; Peprotech) and 1% penicillin/streptomycin. After 7 days, Tie1 expression was analyzed by FACS.
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