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257 nt cf

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The 257-NT/CF is a laboratory equipment product manufactured by R&D Systems. It is designed to perform a core function, but a detailed description while maintaining an unbiased and factual approach is not available.

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Lab products found in correlation

212 gd cf, by R&D Systems (3 mentions) Sb431542, by Bio-Techne (3 mentions) Neurobasal media, by Thermo Fisher Scientific (3 mentions) Fluorodeoxyuridine, by Merck Group (1 mentions) Uridine, by Merck Group (1 mentions) N2 media, by Thermo Fisher Scientific (1 mentions)

4 protocols using 257 nt cf

1

Differentiation of hPSCs into Neurons

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hPSCs were co-infected with TetO-Ngn2-Puro and TetO-GFP (gift from the Wernig Lab) and reverse tetracycline-controlled transactivator (rtTA), and were plated at a density of 100,000 cells/cm2 with rock inhibitor Y27632 (Stemgent 04-0012). Day 1 cells were differentiated in KSR media with 10 μM SB431542 (1614, Tocris), 2 μM XAV939 (04-00046, Stemgent) and 100 nM LDN-193189 (04-0074, Stemgent) along with doxycycline hyclate (2 μg/mL) (maintained for the entire differentiation process, unless noted). Day 2 media were 50% KSR+SB/XAV/LDN and 50% N2 supplemented with puromycin (5 μg/μL), and differentiation media were as previously described (Maroof et al., 2013 (link)). Day 3, N2 media, day 4, neurobasal media (Gibco) supplemented with B27 (50×, Gibco), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CTNF), and glial cell-derived neurotrophic factor (GDNF) (R&D Systems 248-BD/CF, 257-NT/CF, and 212-GD/CF at 10 ng/mL). When co-cultured with glia, day 4 cells were dissociated with accutase and plated at a density of 40,000/cm2 with mouse primary cortical glial cells (seeded at 70,000/cm2 glia) on geltrex-coated plates. Primary glial preparations from post-natal day (P)0–P2 mouse pups were obtained as previously described (Di Giorgio et al., 2008 (link)).
Nehme R., Zuccaro E., Dia Ghosh S., Li C., Sherwood J.L., Pietilainen O., Barrett L.E., Limone F., Worringer K.A., Kommineni S., Zang Y., Cacchiarelli D., Meissner A., Adolfsson R., Haggarty S., Madison J., Muller M., Arlotta P., Fu Z., Feng G, & Eggan K. (2018). Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission. Cell reports, 23(8), 2509-2523.
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2

Differentiation of hPSCs into Neurons

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
hPSCs were co-infected with TetO-Ngn2-Puro and TetO-GFP (gift from the Wernig Lab) and reverse tetracycline-controlled transactivator (rtTA), and were plated at a density of 100,000 cells/cm2 with rock inhibitor Y27632 (Stemgent 04-0012). Day 1 cells were differentiated in KSR media with 10 μM SB431542 (1614, Tocris), 2 μM XAV939 (04-00046, Stemgent) and 100 nM LDN-193189 (04-0074, Stemgent) along with doxycycline hyclate (2 μg/mL) (maintained for the entire differentiation process, unless noted). Day 2 media were 50% KSR+SB/XAV/LDN and 50% N2 supplemented with puromycin (5 μg/μL), and differentiation media were as previously described (Maroof et al., 2013 (link)). Day 3, N2 media, day 4, neurobasal media (Gibco) supplemented with B27 (50×, Gibco), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CTNF), and glial cell-derived neurotrophic factor (GDNF) (R&D Systems 248-BD/CF, 257-NT/CF, and 212-GD/CF at 10 ng/mL). When co-cultured with glia, day 4 cells were dissociated with accutase and plated at a density of 40,000/cm2 with mouse primary cortical glial cells (seeded at 70,000/cm2 glia) on geltrex-coated plates. Primary glial preparations from post-natal day (P)0–P2 mouse pups were obtained as previously described (Di Giorgio et al., 2008 (link)).
Nehme R., Zuccaro E., Dia Ghosh S., Li C., Sherwood J.L., Pietilainen O., Barrett L.E., Limone F., Worringer K.A., Kommineni S., Zang Y., Cacchiarelli D., Meissner A., Adolfsson R., Haggarty S., Madison J., Muller M., Arlotta P., Fu Z., Feng G, & Eggan K. (2018). Combining NGN2 Programming with Developmental Patterning Generates Human Excitatory Neurons with NMDAR-Mediated Synaptic Transmission. Cell reports, 23(8), 2509-2523.
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3

Long-term Motor Neuron Co-culture Protocol

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MN media: neurobasal with 1:50 B27, 1:100 Glutamax, 100 μl 1:100 Beta-mercaptoethanol, 1:50 FBS, 1:100 Pen/Strep. For long-term co-cultures supplemented with GDNF (10 ng/ml, R&D systems 212-GD/CF), BDNF (10 ng/ml, Peprotech 450-02-50UG), CNTF (10 ng/ml, R&D Systems 257-NT/CF), IGF (10 ng/ml, R&D Systems 291-G1-200), 1:5000 UFDU for 1.5 weeks (10 mM each Uridine [Sigma-Aldrich; Catalog Number U3750] and FluorodeoxyUridine [Sigma-Aldrich; Catalog Number F0503]).
Activity experiments: to block activity 1 μM of TTX was added to the MN media from DIV0 to DIV28.
Deriving primary astrocytes: astrocyte-enriched primary cortical glial cultures were acquired from P2 mouse cortices as previously described86 (link). Plates were shaken in 37 °C for 6 h to reduce microglial contamination. Plated astrocytes were passaged once and then frozen in 90% FBS + 10% DMSO.
Deriving Chat-CRE;SUN1-GFP ESC line: Chat-Cre homozygous males were bred to SUN1-GFP homozygous female, and trans-heterozygous blastocysts were collected for stem cell derivation as previously published79 (link).
Patel T., Hammelman J., Aziz S., Jang S., Closser M., Michaels T.L., Blum J.A., Gifford D.K, & Wichterle H. (2022). Transcriptional dynamics of murine motor neuron maturation in vivo and in vitro. Nature Communications, 13, 5427.
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4

Differentiation of iPSCs into Neurons

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iPSCs stably expressing TetO-Ngn2-Neo and reverse tetracycline-controlled transactivator (rtTA) were plated at a density of 40,000 cells cm−2 with rock inhibitor Y27632 (Stemgent, 04-0012). Day 1 cells were differentiated in N2 media (Life Technologies) supplemented with 10 μM SB431542 (Tocris, 1614), 2 μM XAV939 (Stemgent, 04-00046) and 100 nM LDN-193189 (Stemgent, 04-0074) along with doxycycline hyclate (2 μg mL−1). Day 2 media was N2+SB/XAV/LDN/doxycycline hyclate and differentiation media were as previously described. On day 3, cell differentiation was continued in neurobasal media (Life Technologies) supplemented with B27 (50X, Thermo Scientific), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CTNF), glial cell-derived neurotrophic factor (GDNF) (R&D Systems 248-BD/CF, 257-NT/CF, and 212-GD/CF at 10 ng mL−1) and doxycyclinehyclate (2 μg mL−1).
Pintacuda G., Hsu Y.H., Tsafou K., Li K.W., Martín J.M., Riseman J., Biagini J.C., Ching J.K., Mena D., Gonzalez-Lozano M.A., Egri S.B., Jaffe J., Smit A.B., Fornelos N., Eggan K.C, & Lage K. (2023). Protein interaction studies in human induced neurons indicate convergent biology underlying autism spectrum disorders. Cell Genomics, 3(3), 100250.
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