Human iPSCs were reprogrammed using the four Yamanaka factors transferred using the Cyto-Tune Sendai reprogramming kit (Invitrogen) or standard retroviral vectors as previously described,21 (link) and cultured as colonies on Geltrex (Thermo Fisher)-coated plates in iPS-Brew (Miltenyi Biotec). For iPSC-iN transgene delivery, iPSCs were transferred to a monolayer PluriPro (Cell Guidance Systems) condition, transduced with lentiviral particles for pLVX-UbC-rtTA-Ngn2:2A:EGFP (UNG), and expanded in the presence of puromycin (0.5 mg/mL; Sigma Aldrich) as ‘iPSC-iN-ready’ iPSC-UNG lines as previously described.76 (link) To initiate conversion, confluent iPSC-UNG monolayer cultures were transferred into NC media to achieve neuronal conversion of the cultures within 3 weeks before fixing and staining, or isolation by flow cytometry for RNA purification.
Pluripro
Manufactured by Cell Guidance Systems
Pluripro is a laboratory equipment product offered by Cell Guidance Systems. It is designed to facilitate the culture and expansion of pluripotent stem cells. The core function of Pluripro is to provide a controlled and optimized environment for the growth and maintenance of pluripotent stem cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Cytotune sendai reprogramming kit, by Thermo Fisher Scientific (2 mentions)
Geltrex, by Thermo Fisher Scientific (2 mentions)
Puromycin, by Merck Group (2 mentions)
Ips brew, by Miltenyi Biotec (2 mentions)
Tryple, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Pencillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Hesc qualified matrigel, by BD (1 mentions)
5 protocols using pluripro
1
Generation of induced neuronal cells from human iPSCs
2
Primary Neuronal Culture from Mouse Brain
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Cultures were prepared from JNPL3 pups at postnatal day 0 as previously described. Briefly, plates were coated with Pluripro (Cell Guidance Systems) for at least 3 h. The cortex and hippocampus of each mouse was removed and washed in modified Hank's Balanced Salt Solution. Tissue was incubated with 0.5% trypsin for 20 min at 37°C, and then subjected to further washing and manual dissociation. Cells to be used for the mixed cortical cultures were maintained in plating media containing glucose and serum. To remove the glia and produce neuronal cultures, the plating media was removed after the first 24 h and replaced with neurobasal media.
3
Characterization of HEK293 and hiPS Cells
4
Efficient Generation of iPSC-Derived Neurons
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Human iPSCs were reprogrammed using the four Yamanaka factors transferred using the Cyto-Tune Sendai reprogramming kit (Invitrogen) or standard retroviral vectors. Based on clonal characterizations (Israel et al., 2012 (link); Young et al., 2015 (link)), one iPSC clone for each of the 21 subjects was selected and cultured as colonies on Geltrex (Thermo Fisher)-coated plates in iPS-Brew (Miltenyi Biotec). For iPSC-iN transgene delivery, iPSCs were transferred to a monolayer PluriPro (Cell Guidance Systems) condition, transduced with lentiviral particles for pLVX-UbC-rtTA-Ngn2:2A:EGFP (UNG; Figure S1 ), and expanded in the presence of puromycin (0.5 μg/ml; Sigma Aldrich) as ‘iPSC-iN-ready’ iPSC-UNG lines as previously described (Schafer et al., 2019 (link)). To initiate conversion, confluent iPSC-UNG monolayer cultures were transferred into NC media to achieve neuronal conversion of the cultures within 3 weeks before fixing and staining, or isolation by flow cytometry for RNA purification.
5
Neuronal Culture from Transgenic Murine Model
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Primary neuronal cultures were prepared from M83 mouse pups at postnatal day 0 according to established protocols (39 (link)). The homozygous M83 mice express the A53T mutation of human α-syn via the mouse prion promoter at approximately twelve times the level of endogenous mouse α-syn. This model develops extensive α-syn inclusions with age, particularly in the homozygous mice at the age used here (5–8 months), and various behavioral deficits have been reported in this model (51 (link)). In brief, plates were coated with Pluripro (Cell Guidance Systems) for at least 3 h. The cortex and hippocampus of each mouse were collected and washed in modified Hank’s Balanced Salt Solution. Tissue was then incubated with 0.5% trypsin for 20 min at 37°C, followed by further washing and manual dissociation. Cells were maintained in plating media, whereas the plating media was replaced with neurobasal media to eliminate glia and produce neuronal cultures after the first 24 h.
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