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212 gd cf

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The 212-GD/CF is a laboratory equipment product designed for research and development purposes. Its core function is to perform cell culture-related tasks, but a detailed description of its intended use cannot be provided in an unbiased and factual manner while maintaining conciseness.

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Lab products found in correlation

Neurobasal media, by Thermo Fisher Scientific (5 mentions) Sb431542, by Bio-Techne (4 mentions) 257 nt cf, by R&D Systems (3 mentions) Doxycycline hyclate, by Thermo Fisher Scientific (1 mentions) 5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (1 mentions) N2 media, by Thermo Fisher Scientific (1 mentions)

5 protocols using 212 gd cf

1

Glutamatergic Neuron Differentiation from ESCs

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Neuronal differentiation of ESCs into cortical glutamatergic neurons was carried out as previously described92 (link). In brief, the differentiation was carried out by adding doxycycline hyclate (2 μg/mL) to N2 supplemented media (Thermo Fisher, 17502048) with patterning factors SB431542 (Tocris, 1614, 10 μM), XAV939 (Stemgent, 04–00046, 2 μM) and LDN-193189 (Stemgent, 04–0074, 100 nM), as described previously92 (link)–94 (link). Puromycin selection was used (5μg/μL), from days 2 to 6 to remove non-transduced cells. At 4 days post induction, neuronal cells were resuspended into Neurobasal media (Gibco, 21103049) that was supplemented with B27 (Gibco, 17504044, 50X), doxycycline (2 μg/mL), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CTNF), and glial cell-derived neurotrophic factor (GDNF) (R&D Systems 248-BD/CF, 257–33 NT/CF, and 212-GD/CF at 10 ng/mL each). From this point onwards the neurons were either co-cultured with murine glial cells that were derived from early postnatal (P1-P3) mouse brains as described previously95 (link) or were left to grow as monocultures (mouse strain https://www.jax.org/strain/100012; animal ethical committee approval by Harvard University: Animal Experimentation Protocol (AEP) # 93–15).
Gazestani V., Kamath T., Nadaf N.M., Burris S., Rooney B., Junkkari A., Vanderburg C., Rauramaa T., Therrien M., Tegtmeyer M., Herukka S.K., Abdulraouf A., Marsh S., Malm T., Hiltunen M., Nehme R., Stevens B., Leinonen V, & Macosko E.Z. (2023). Early Alzheimer’s disease pathology in human cortex is associated with a transient phase of distinct cell states. bioRxiv.
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2

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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3

Differentiation of hPSCs into Neurons

Cited 1 time
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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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4

Generation of Neuronal Cell Villages

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The cell villages were generated as described previously.27 (link),28 (link) At day 3 of the differentiation, the differentiating cells from 48 donors were passaged into differentiation media that was supplemented with 5-Ethynyl-2’-deoxyuridine (Life Technologies, A10044, 10 μM). Cell villages of the immature neurons were generated at day 6 by dissociating the cells with Accutase®, counting them by the Scepter™ Automated Cell Counter (Millipore Sigma) and plating the cells at a density of 40 000 cells/cm2 in Neurobasal media (Gibco, 21103049) supplemented with B27 (Gibco, 17504044, 50X), doxycycline (2 μg/mL), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CTNF), and glial cell-derived neurotrophic factor (GDNF) (R&D Systems 248-BD/CF, 257-33 NT/CF, and 212-GD/CF at 10 ng/mL each). Neuronal villages were either grown as monocultures or co-cultured with murine glial cells (at a density of 70 000 cells/cm2). Villages were harvested for single cell RNA sequencing at day 28 of the differentiation.
, & Igloi G.L. (1998). Variability in the stability of DNA–peptide nucleic acid (PNA) single-base mismatched duplexes: Real-time hybridization during affinity electrophoresis in PNA-containing gels. Proceedings of the National Academy of Sciences of the United States of America, 95(15), 8562-8567.
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5

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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