dopaminergic neuron differentiation medium Search Results


dmem f12 containing dopaminergic neuron differentiation kit  (ATCC)
94
ATCC dmem f12 containing dopaminergic neuron differentiation kit
Dmem F12 Containing Dopaminergic Neuron Differentiation Kit, supplied by ATCC, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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human dopaminergic neuron maturation medium  (iXCells Biotechnologies)
93
iXCells Biotechnologies human dopaminergic neuron maturation medium
Human Dopaminergic Neuron Maturation Medium, supplied by iXCells Biotechnologies, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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™ psc dopaminergic neuron differentiation kit  (Thermo Fisher)
90
Thermo Fisher ™ psc dopaminergic neuron differentiation kit
™ Psc Dopaminergic Neuron Differentiation Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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™ psc dopaminergic neuron differentiation kit - by Bioz Stars, 2026-02
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brainphys neuronal medium  (STEMCELL Technologies Inc)
90
STEMCELL Technologies Inc brainphys neuronal medium
Brainphys Neuronal Medium, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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brainphys neuronal medium stemcell 05790  (STEMCELL Technologies Inc)
90
STEMCELL Technologies Inc brainphys neuronal medium stemcell 05790
( A ) Sonication of al α-syn fibrils results in significantly smaller fibril length, allowing the α-syn oligomers to be characterized as PFF with a length of below 100 nm. ( B ) The mean length of samples used includes 51.72, 52.89, 58.28, 67.95, 95.04 nm. Once confirmed with electron microscopy, the corresponding batches were tagged with Alexa Fluor 488, Alexa Fluor 633, or 5 nm gold ( C ). Scale Bar = 100 nm. (D) Trypan blue exclusion assay used to discriminate against intra- and extracellular PFF. HeLa cells were grown on glass-bottomed plates and placed on ice for 30 min. They were then transferred to an imaging chamber chamber for live imaging. For the top two rows, (4°C), the chamber was not heated with the cells remaining at low temperature. Images were taken from before and after PFF addition. Trypan blue was added to cells 2 min following PFF addition. No PFF was internalized in this condition, as trypan blue was able to quench all PFF fluorescence. For the bottom two rows (37°C), the chamber was heated to 37°C prior to the addition of PFF. PFF was then added, and 2 min following PFF addition, trypan blue was added. PFF fluorescence not quenched by trypan blue is internalized PFF. Scale bar = 20 µm for low magnification and 5 µm for insets. ( E ) PFF was added to cells at 4°C for 30 min to allow the binding of PFF to the cell surface without internalization. Cells were then transferred to a pre-heated live imaging chamber. Arrows point to specific PFF punctae that colocalize with lysosomes over time. Scale bar = 10 µm for the low magnification image and 5 µm for the insets. ( F ) Unlike cargo proteins like Tf and EGF, trypsinization for 90 sec on ice is needed to remove extracellular PFF. In this experiment, Cells were incubated with PFF for 30 min at 4°C. Since PFF internalization is temperature dependent, we expected no PFF internalization. We then assessed the efficacy of PBS, Acid, and Trypsin wash on their ability to remove extracellular PFF. We found that trypsinization was the only method that removes extracellular PFF. Scale bar = 20 µm. ( G ) To further confirm the efficacy of trypsin wash for the removal of extracellular PFF, PBS (control) or PFF were added to cells. Cells were incubated at 4°C for 30 min. Cells exposed to PFF were then washed with either PBS or with trypsin. Cell lysates were then immunoblotted for α-syn, to assess the presence of PFF. Samples washed with trypsin showed no α-syn signal, while the cells washed with PBS did, confirming the efficacy of trypsin in removing extracellular PFF. ( H ) To confirm the identity of astrocytes, glioblastoma cell lines, and the iPSC derived neural progenitor cells along with differentiated neurons, marker antibodies were used. Astrocytic and glial identity was confirmed using the glial fibrillary associated protein (GFAP) antibody. As expected, fetal astrocytes, and U251 had high levels of GFAP fluorescence while U343 had less, and U87 do not show any GFAP fluorescence. For neurons, neuronal markers such as microtubule-associated protein 2 (MAP2) was used. To confirm the identity of <t>dopaminergic</t> neurons, tyrosine hydroxylase antibody (TH) was used. The confirm the identity of neural progenitor cells beta-III tubulin was used. Finally, to confirm the identity of dopaminergic neural progenitor cells, Forkhead box protein A2 (FOXA2) was used. Scale bar = 20 µm.
Brainphys Neuronal Medium Stemcell 05790, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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n2 supplement  (Thermo Fisher)
90
Thermo Fisher n2 supplement
( A ) Sonication of al α-syn fibrils results in significantly smaller fibril length, allowing the α-syn oligomers to be characterized as PFF with a length of below 100 nm. ( B ) The mean length of samples used includes 51.72, 52.89, 58.28, 67.95, 95.04 nm. Once confirmed with electron microscopy, the corresponding batches were tagged with Alexa Fluor 488, Alexa Fluor 633, or 5 nm gold ( C ). Scale Bar = 100 nm. (D) Trypan blue exclusion assay used to discriminate against intra- and extracellular PFF. HeLa cells were grown on glass-bottomed plates and placed on ice for 30 min. They were then transferred to an imaging chamber chamber for live imaging. For the top two rows, (4°C), the chamber was not heated with the cells remaining at low temperature. Images were taken from before and after PFF addition. Trypan blue was added to cells 2 min following PFF addition. No PFF was internalized in this condition, as trypan blue was able to quench all PFF fluorescence. For the bottom two rows (37°C), the chamber was heated to 37°C prior to the addition of PFF. PFF was then added, and 2 min following PFF addition, trypan blue was added. PFF fluorescence not quenched by trypan blue is internalized PFF. Scale bar = 20 µm for low magnification and 5 µm for insets. ( E ) PFF was added to cells at 4°C for 30 min to allow the binding of PFF to the cell surface without internalization. Cells were then transferred to a pre-heated live imaging chamber. Arrows point to specific PFF punctae that colocalize with lysosomes over time. Scale bar = 10 µm for the low magnification image and 5 µm for the insets. ( F ) Unlike cargo proteins like Tf and EGF, trypsinization for 90 sec on ice is needed to remove extracellular PFF. In this experiment, Cells were incubated with PFF for 30 min at 4°C. Since PFF internalization is temperature dependent, we expected no PFF internalization. We then assessed the efficacy of PBS, Acid, and Trypsin wash on their ability to remove extracellular PFF. We found that trypsinization was the only method that removes extracellular PFF. Scale bar = 20 µm. ( G ) To further confirm the efficacy of trypsin wash for the removal of extracellular PFF, PBS (control) or PFF were added to cells. Cells were incubated at 4°C for 30 min. Cells exposed to PFF were then washed with either PBS or with trypsin. Cell lysates were then immunoblotted for α-syn, to assess the presence of PFF. Samples washed with trypsin showed no α-syn signal, while the cells washed with PBS did, confirming the efficacy of trypsin in removing extracellular PFF. ( H ) To confirm the identity of astrocytes, glioblastoma cell lines, and the iPSC derived neural progenitor cells along with differentiated neurons, marker antibodies were used. Astrocytic and glial identity was confirmed using the glial fibrillary associated protein (GFAP) antibody. As expected, fetal astrocytes, and U251 had high levels of GFAP fluorescence while U343 had less, and U87 do not show any GFAP fluorescence. For neurons, neuronal markers such as microtubule-associated protein 2 (MAP2) was used. To confirm the identity of <t>dopaminergic</t> neurons, tyrosine hydroxylase antibody (TH) was used. The confirm the identity of neural progenitor cells beta-III tubulin was used. Finally, to confirm the identity of dopaminergic neural progenitor cells, Forkhead box protein A2 (FOXA2) was used. Scale bar = 20 µm.
N2 Supplement, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/n2 supplement/product/Thermo Fisher
Average 90 stars, based on 1 article reviews
n2 supplement - by Bioz Stars, 2026-02
90/100 stars
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dopamine neuron medium (dpm)  (Thermo Fisher)
90
Thermo Fisher dopamine neuron medium (dpm)
( A ) Sonication of al α-syn fibrils results in significantly smaller fibril length, allowing the α-syn oligomers to be characterized as PFF with a length of below 100 nm. ( B ) The mean length of samples used includes 51.72, 52.89, 58.28, 67.95, 95.04 nm. Once confirmed with electron microscopy, the corresponding batches were tagged with Alexa Fluor 488, Alexa Fluor 633, or 5 nm gold ( C ). Scale Bar = 100 nm. (D) Trypan blue exclusion assay used to discriminate against intra- and extracellular PFF. HeLa cells were grown on glass-bottomed plates and placed on ice for 30 min. They were then transferred to an imaging chamber chamber for live imaging. For the top two rows, (4°C), the chamber was not heated with the cells remaining at low temperature. Images were taken from before and after PFF addition. Trypan blue was added to cells 2 min following PFF addition. No PFF was internalized in this condition, as trypan blue was able to quench all PFF fluorescence. For the bottom two rows (37°C), the chamber was heated to 37°C prior to the addition of PFF. PFF was then added, and 2 min following PFF addition, trypan blue was added. PFF fluorescence not quenched by trypan blue is internalized PFF. Scale bar = 20 µm for low magnification and 5 µm for insets. ( E ) PFF was added to cells at 4°C for 30 min to allow the binding of PFF to the cell surface without internalization. Cells were then transferred to a pre-heated live imaging chamber. Arrows point to specific PFF punctae that colocalize with lysosomes over time. Scale bar = 10 µm for the low magnification image and 5 µm for the insets. ( F ) Unlike cargo proteins like Tf and EGF, trypsinization for 90 sec on ice is needed to remove extracellular PFF. In this experiment, Cells were incubated with PFF for 30 min at 4°C. Since PFF internalization is temperature dependent, we expected no PFF internalization. We then assessed the efficacy of PBS, Acid, and Trypsin wash on their ability to remove extracellular PFF. We found that trypsinization was the only method that removes extracellular PFF. Scale bar = 20 µm. ( G ) To further confirm the efficacy of trypsin wash for the removal of extracellular PFF, PBS (control) or PFF were added to cells. Cells were incubated at 4°C for 30 min. Cells exposed to PFF were then washed with either PBS or with trypsin. Cell lysates were then immunoblotted for α-syn, to assess the presence of PFF. Samples washed with trypsin showed no α-syn signal, while the cells washed with PBS did, confirming the efficacy of trypsin in removing extracellular PFF. ( H ) To confirm the identity of astrocytes, glioblastoma cell lines, and the iPSC derived neural progenitor cells along with differentiated neurons, marker antibodies were used. Astrocytic and glial identity was confirmed using the glial fibrillary associated protein (GFAP) antibody. As expected, fetal astrocytes, and U251 had high levels of GFAP fluorescence while U343 had less, and U87 do not show any GFAP fluorescence. For neurons, neuronal markers such as microtubule-associated protein 2 (MAP2) was used. To confirm the identity of <t>dopaminergic</t> neurons, tyrosine hydroxylase antibody (TH) was used. The confirm the identity of neural progenitor cells beta-III tubulin was used. Finally, to confirm the identity of dopaminergic neural progenitor cells, Forkhead box protein A2 (FOXA2) was used. Scale bar = 20 µm.
Dopamine Neuron Medium (Dpm), supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/dopamine neuron medium (dpm)/product/Thermo Fisher
Average 90 stars, based on 1 article reviews
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stemdifftm dopaminergic neuron maturation medium 1  (STEMCELL Technologies Inc)
90
STEMCELL Technologies Inc stemdifftm dopaminergic neuron maturation medium 1
( A ) Sonication of al α-syn fibrils results in significantly smaller fibril length, allowing the α-syn oligomers to be characterized as PFF with a length of below 100 nm. ( B ) The mean length of samples used includes 51.72, 52.89, 58.28, 67.95, 95.04 nm. Once confirmed with electron microscopy, the corresponding batches were tagged with Alexa Fluor 488, Alexa Fluor 633, or 5 nm gold ( C ). Scale Bar = 100 nm. (D) Trypan blue exclusion assay used to discriminate against intra- and extracellular PFF. HeLa cells were grown on glass-bottomed plates and placed on ice for 30 min. They were then transferred to an imaging chamber chamber for live imaging. For the top two rows, (4°C), the chamber was not heated with the cells remaining at low temperature. Images were taken from before and after PFF addition. Trypan blue was added to cells 2 min following PFF addition. No PFF was internalized in this condition, as trypan blue was able to quench all PFF fluorescence. For the bottom two rows (37°C), the chamber was heated to 37°C prior to the addition of PFF. PFF was then added, and 2 min following PFF addition, trypan blue was added. PFF fluorescence not quenched by trypan blue is internalized PFF. Scale bar = 20 µm for low magnification and 5 µm for insets. ( E ) PFF was added to cells at 4°C for 30 min to allow the binding of PFF to the cell surface without internalization. Cells were then transferred to a pre-heated live imaging chamber. Arrows point to specific PFF punctae that colocalize with lysosomes over time. Scale bar = 10 µm for the low magnification image and 5 µm for the insets. ( F ) Unlike cargo proteins like Tf and EGF, trypsinization for 90 sec on ice is needed to remove extracellular PFF. In this experiment, Cells were incubated with PFF for 30 min at 4°C. Since PFF internalization is temperature dependent, we expected no PFF internalization. We then assessed the efficacy of PBS, Acid, and Trypsin wash on their ability to remove extracellular PFF. We found that trypsinization was the only method that removes extracellular PFF. Scale bar = 20 µm. ( G ) To further confirm the efficacy of trypsin wash for the removal of extracellular PFF, PBS (control) or PFF were added to cells. Cells were incubated at 4°C for 30 min. Cells exposed to PFF were then washed with either PBS or with trypsin. Cell lysates were then immunoblotted for α-syn, to assess the presence of PFF. Samples washed with trypsin showed no α-syn signal, while the cells washed with PBS did, confirming the efficacy of trypsin in removing extracellular PFF. ( H ) To confirm the identity of astrocytes, glioblastoma cell lines, and the iPSC derived neural progenitor cells along with differentiated neurons, marker antibodies were used. Astrocytic and glial identity was confirmed using the glial fibrillary associated protein (GFAP) antibody. As expected, fetal astrocytes, and U251 had high levels of GFAP fluorescence while U343 had less, and U87 do not show any GFAP fluorescence. For neurons, neuronal markers such as microtubule-associated protein 2 (MAP2) was used. To confirm the identity of <t>dopaminergic</t> neurons, tyrosine hydroxylase antibody (TH) was used. The confirm the identity of neural progenitor cells beta-III tubulin was used. Finally, to confirm the identity of dopaminergic neural progenitor cells, Forkhead box protein A2 (FOXA2) was used. Scale bar = 20 µm.
Stemdifftm Dopaminergic Neuron Maturation Medium 1, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/stemdifftm dopaminergic neuron maturation medium 1/product/STEMCELL Technologies Inc
Average 90 stars, based on 1 article reviews
stemdifftm dopaminergic neuron maturation medium 1 - by Bioz Stars, 2026-02
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stemdiff dopaminergic neuron differentiation kit  (STEMCELL Technologies Inc)
90
STEMCELL Technologies Inc stemdiff dopaminergic neuron differentiation kit
( A ) Sonication of al α-syn fibrils results in significantly smaller fibril length, allowing the α-syn oligomers to be characterized as PFF with a length of below 100 nm. ( B ) The mean length of samples used includes 51.72, 52.89, 58.28, 67.95, 95.04 nm. Once confirmed with electron microscopy, the corresponding batches were tagged with Alexa Fluor 488, Alexa Fluor 633, or 5 nm gold ( C ). Scale Bar = 100 nm. (D) Trypan blue exclusion assay used to discriminate against intra- and extracellular PFF. HeLa cells were grown on glass-bottomed plates and placed on ice for 30 min. They were then transferred to an imaging chamber chamber for live imaging. For the top two rows, (4°C), the chamber was not heated with the cells remaining at low temperature. Images were taken from before and after PFF addition. Trypan blue was added to cells 2 min following PFF addition. No PFF was internalized in this condition, as trypan blue was able to quench all PFF fluorescence. For the bottom two rows (37°C), the chamber was heated to 37°C prior to the addition of PFF. PFF was then added, and 2 min following PFF addition, trypan blue was added. PFF fluorescence not quenched by trypan blue is internalized PFF. Scale bar = 20 µm for low magnification and 5 µm for insets. ( E ) PFF was added to cells at 4°C for 30 min to allow the binding of PFF to the cell surface without internalization. Cells were then transferred to a pre-heated live imaging chamber. Arrows point to specific PFF punctae that colocalize with lysosomes over time. Scale bar = 10 µm for the low magnification image and 5 µm for the insets. ( F ) Unlike cargo proteins like Tf and EGF, trypsinization for 90 sec on ice is needed to remove extracellular PFF. In this experiment, Cells were incubated with PFF for 30 min at 4°C. Since PFF internalization is temperature dependent, we expected no PFF internalization. We then assessed the efficacy of PBS, Acid, and Trypsin wash on their ability to remove extracellular PFF. We found that trypsinization was the only method that removes extracellular PFF. Scale bar = 20 µm. ( G ) To further confirm the efficacy of trypsin wash for the removal of extracellular PFF, PBS (control) or PFF were added to cells. Cells were incubated at 4°C for 30 min. Cells exposed to PFF were then washed with either PBS or with trypsin. Cell lysates were then immunoblotted for α-syn, to assess the presence of PFF. Samples washed with trypsin showed no α-syn signal, while the cells washed with PBS did, confirming the efficacy of trypsin in removing extracellular PFF. ( H ) To confirm the identity of astrocytes, glioblastoma cell lines, and the iPSC derived neural progenitor cells along with differentiated neurons, marker antibodies were used. Astrocytic and glial identity was confirmed using the glial fibrillary associated protein (GFAP) antibody. As expected, fetal astrocytes, and U251 had high levels of GFAP fluorescence while U343 had less, and U87 do not show any GFAP fluorescence. For neurons, neuronal markers such as microtubule-associated protein 2 (MAP2) was used. To confirm the identity of <t>dopaminergic</t> neurons, tyrosine hydroxylase antibody (TH) was used. The confirm the identity of neural progenitor cells beta-III tubulin was used. Finally, to confirm the identity of dopaminergic neural progenitor cells, Forkhead box protein A2 (FOXA2) was used. Scale bar = 20 µm.
Stemdiff Dopaminergic Neuron Differentiation Kit, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/stemdiff dopaminergic neuron differentiation kit/product/STEMCELL Technologies Inc
Average 90 stars, based on 1 article reviews
stemdiff dopaminergic neuron differentiation kit - by Bioz Stars, 2026-02
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dopaminergic neuron maturation medium  (Thermo Fisher)
90
Thermo Fisher dopaminergic neuron maturation medium
( A ) Sonication of al α-syn fibrils results in significantly smaller fibril length, allowing the α-syn oligomers to be characterized as PFF with a length of below 100 nm. ( B ) The mean length of samples used includes 51.72, 52.89, 58.28, 67.95, 95.04 nm. Once confirmed with electron microscopy, the corresponding batches were tagged with Alexa Fluor 488, Alexa Fluor 633, or 5 nm gold ( C ). Scale Bar = 100 nm. (D) Trypan blue exclusion assay used to discriminate against intra- and extracellular PFF. HeLa cells were grown on glass-bottomed plates and placed on ice for 30 min. They were then transferred to an imaging chamber chamber for live imaging. For the top two rows, (4°C), the chamber was not heated with the cells remaining at low temperature. Images were taken from before and after PFF addition. Trypan blue was added to cells 2 min following PFF addition. No PFF was internalized in this condition, as trypan blue was able to quench all PFF fluorescence. For the bottom two rows (37°C), the chamber was heated to 37°C prior to the addition of PFF. PFF was then added, and 2 min following PFF addition, trypan blue was added. PFF fluorescence not quenched by trypan blue is internalized PFF. Scale bar = 20 µm for low magnification and 5 µm for insets. ( E ) PFF was added to cells at 4°C for 30 min to allow the binding of PFF to the cell surface without internalization. Cells were then transferred to a pre-heated live imaging chamber. Arrows point to specific PFF punctae that colocalize with lysosomes over time. Scale bar = 10 µm for the low magnification image and 5 µm for the insets. ( F ) Unlike cargo proteins like Tf and EGF, trypsinization for 90 sec on ice is needed to remove extracellular PFF. In this experiment, Cells were incubated with PFF for 30 min at 4°C. Since PFF internalization is temperature dependent, we expected no PFF internalization. We then assessed the efficacy of PBS, Acid, and Trypsin wash on their ability to remove extracellular PFF. We found that trypsinization was the only method that removes extracellular PFF. Scale bar = 20 µm. ( G ) To further confirm the efficacy of trypsin wash for the removal of extracellular PFF, PBS (control) or PFF were added to cells. Cells were incubated at 4°C for 30 min. Cells exposed to PFF were then washed with either PBS or with trypsin. Cell lysates were then immunoblotted for α-syn, to assess the presence of PFF. Samples washed with trypsin showed no α-syn signal, while the cells washed with PBS did, confirming the efficacy of trypsin in removing extracellular PFF. ( H ) To confirm the identity of astrocytes, glioblastoma cell lines, and the iPSC derived neural progenitor cells along with differentiated neurons, marker antibodies were used. Astrocytic and glial identity was confirmed using the glial fibrillary associated protein (GFAP) antibody. As expected, fetal astrocytes, and U251 had high levels of GFAP fluorescence while U343 had less, and U87 do not show any GFAP fluorescence. For neurons, neuronal markers such as microtubule-associated protein 2 (MAP2) was used. To confirm the identity of <t>dopaminergic</t> neurons, tyrosine hydroxylase antibody (TH) was used. The confirm the identity of neural progenitor cells beta-III tubulin was used. Finally, to confirm the identity of dopaminergic neural progenitor cells, Forkhead box protein A2 (FOXA2) was used. Scale bar = 20 µm.
Dopaminergic Neuron Maturation Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/dopaminergic neuron maturation medium/product/Thermo Fisher
Average 90 stars, based on 1 article reviews
dopaminergic neuron maturation medium - by Bioz Stars, 2026-02
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dopaminergic neuron maintenance medium  (Axol Bioscience)
N/A
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Image Search Results


( A ) Sonication of al α-syn fibrils results in significantly smaller fibril length, allowing the α-syn oligomers to be characterized as PFF with a length of below 100 nm. ( B ) The mean length of samples used includes 51.72, 52.89, 58.28, 67.95, 95.04 nm. Once confirmed with electron microscopy, the corresponding batches were tagged with Alexa Fluor 488, Alexa Fluor 633, or 5 nm gold ( C ). Scale Bar = 100 nm. (D) Trypan blue exclusion assay used to discriminate against intra- and extracellular PFF. HeLa cells were grown on glass-bottomed plates and placed on ice for 30 min. They were then transferred to an imaging chamber chamber for live imaging. For the top two rows, (4°C), the chamber was not heated with the cells remaining at low temperature. Images were taken from before and after PFF addition. Trypan blue was added to cells 2 min following PFF addition. No PFF was internalized in this condition, as trypan blue was able to quench all PFF fluorescence. For the bottom two rows (37°C), the chamber was heated to 37°C prior to the addition of PFF. PFF was then added, and 2 min following PFF addition, trypan blue was added. PFF fluorescence not quenched by trypan blue is internalized PFF. Scale bar = 20 µm for low magnification and 5 µm for insets. ( E ) PFF was added to cells at 4°C for 30 min to allow the binding of PFF to the cell surface without internalization. Cells were then transferred to a pre-heated live imaging chamber. Arrows point to specific PFF punctae that colocalize with lysosomes over time. Scale bar = 10 µm for the low magnification image and 5 µm for the insets. ( F ) Unlike cargo proteins like Tf and EGF, trypsinization for 90 sec on ice is needed to remove extracellular PFF. In this experiment, Cells were incubated with PFF for 30 min at 4°C. Since PFF internalization is temperature dependent, we expected no PFF internalization. We then assessed the efficacy of PBS, Acid, and Trypsin wash on their ability to remove extracellular PFF. We found that trypsinization was the only method that removes extracellular PFF. Scale bar = 20 µm. ( G ) To further confirm the efficacy of trypsin wash for the removal of extracellular PFF, PBS (control) or PFF were added to cells. Cells were incubated at 4°C for 30 min. Cells exposed to PFF were then washed with either PBS or with trypsin. Cell lysates were then immunoblotted for α-syn, to assess the presence of PFF. Samples washed with trypsin showed no α-syn signal, while the cells washed with PBS did, confirming the efficacy of trypsin in removing extracellular PFF. ( H ) To confirm the identity of astrocytes, glioblastoma cell lines, and the iPSC derived neural progenitor cells along with differentiated neurons, marker antibodies were used. Astrocytic and glial identity was confirmed using the glial fibrillary associated protein (GFAP) antibody. As expected, fetal astrocytes, and U251 had high levels of GFAP fluorescence while U343 had less, and U87 do not show any GFAP fluorescence. For neurons, neuronal markers such as microtubule-associated protein 2 (MAP2) was used. To confirm the identity of dopaminergic neurons, tyrosine hydroxylase antibody (TH) was used. The confirm the identity of neural progenitor cells beta-III tubulin was used. Finally, to confirm the identity of dopaminergic neural progenitor cells, Forkhead box protein A2 (FOXA2) was used. Scale bar = 20 µm.

Journal: bioRxiv

Article Title: A novel form of macropinocytosis mediates ultra-rapid transfer of pathological alpha-synuclein to lysosomes

doi: 10.1101/2022.01.06.475207

Figure Lengend Snippet: ( A ) Sonication of al α-syn fibrils results in significantly smaller fibril length, allowing the α-syn oligomers to be characterized as PFF with a length of below 100 nm. ( B ) The mean length of samples used includes 51.72, 52.89, 58.28, 67.95, 95.04 nm. Once confirmed with electron microscopy, the corresponding batches were tagged with Alexa Fluor 488, Alexa Fluor 633, or 5 nm gold ( C ). Scale Bar = 100 nm. (D) Trypan blue exclusion assay used to discriminate against intra- and extracellular PFF. HeLa cells were grown on glass-bottomed plates and placed on ice for 30 min. They were then transferred to an imaging chamber chamber for live imaging. For the top two rows, (4°C), the chamber was not heated with the cells remaining at low temperature. Images were taken from before and after PFF addition. Trypan blue was added to cells 2 min following PFF addition. No PFF was internalized in this condition, as trypan blue was able to quench all PFF fluorescence. For the bottom two rows (37°C), the chamber was heated to 37°C prior to the addition of PFF. PFF was then added, and 2 min following PFF addition, trypan blue was added. PFF fluorescence not quenched by trypan blue is internalized PFF. Scale bar = 20 µm for low magnification and 5 µm for insets. ( E ) PFF was added to cells at 4°C for 30 min to allow the binding of PFF to the cell surface without internalization. Cells were then transferred to a pre-heated live imaging chamber. Arrows point to specific PFF punctae that colocalize with lysosomes over time. Scale bar = 10 µm for the low magnification image and 5 µm for the insets. ( F ) Unlike cargo proteins like Tf and EGF, trypsinization for 90 sec on ice is needed to remove extracellular PFF. In this experiment, Cells were incubated with PFF for 30 min at 4°C. Since PFF internalization is temperature dependent, we expected no PFF internalization. We then assessed the efficacy of PBS, Acid, and Trypsin wash on their ability to remove extracellular PFF. We found that trypsinization was the only method that removes extracellular PFF. Scale bar = 20 µm. ( G ) To further confirm the efficacy of trypsin wash for the removal of extracellular PFF, PBS (control) or PFF were added to cells. Cells were incubated at 4°C for 30 min. Cells exposed to PFF were then washed with either PBS or with trypsin. Cell lysates were then immunoblotted for α-syn, to assess the presence of PFF. Samples washed with trypsin showed no α-syn signal, while the cells washed with PBS did, confirming the efficacy of trypsin in removing extracellular PFF. ( H ) To confirm the identity of astrocytes, glioblastoma cell lines, and the iPSC derived neural progenitor cells along with differentiated neurons, marker antibodies were used. Astrocytic and glial identity was confirmed using the glial fibrillary associated protein (GFAP) antibody. As expected, fetal astrocytes, and U251 had high levels of GFAP fluorescence while U343 had less, and U87 do not show any GFAP fluorescence. For neurons, neuronal markers such as microtubule-associated protein 2 (MAP2) was used. To confirm the identity of dopaminergic neurons, tyrosine hydroxylase antibody (TH) was used. The confirm the identity of neural progenitor cells beta-III tubulin was used. Finally, to confirm the identity of dopaminergic neural progenitor cells, Forkhead box protein A2 (FOXA2) was used. Scale bar = 20 µm.

Article Snippet: To further differentiate into dopaminergic neurons, neural progenitor medium was switched to dopaminergic neural differentiation medium (Brainphys Neuronal medium, STEMCELL Technologies 05790) supplemented with N2A Supplement A (STEMCELL Technologies; 07152), Neurocult SM1 Neuronal Supplement (STEMCELL Technologies; 05711), BDNF (20 ng/mL; Peprotech; 450-02), GDNF (20 ng/mL; Peprotech; 450-10), Compound E (0.1 μM; STEMCELL Technologies; 73954), db-cAMP (0.5 mM; Carbosynth; ND07996), Ascorbic acid (200 μM; Sigma; A5960) and laminin (1 μg/mL, Sigma; L2020).

Techniques: Sonication, Electron Microscopy, Trypan Blue Exclusion Assay, Imaging, Fluorescence, Binding Assay, Incubation, Control, Derivative Assay, Marker

( A ) Dopaminergic NPCs were incubated for 0, 2, 10, 30 min at 37°C following addition of Alex488-labelled PFF at 2 µg/mL. The cells were then washed with trypsin, fixed, and stained with lysotracker. Scale bar = 20 µm. ( B ) NPCs differentiated into dopaminergic neurons were processed as in A . Scale bar = 20 µm. ( C ) Human astrocytes were grown and mounted on coverslips. PFF was added to each coverslip at 1 µg/ml. Cells were incubated for 0, 2, 10, 30 min at 37°C following addition of Alex488-labelled PFF. Cells were washed with trypsin, fixed, permeabilized, and stained with LAMP1. Scale bar = 20 µm and 2.5 µm insets. ( D/E ) Colocalization of Lysotracker with PFF from experiments as in A and B. n = 6 for neurons and n = 6 for NPCs in each condition (i.e., n = 48 total), performed in three independent experiments. ( F ) Colocalization rate of LAMP1 with PFF from experiments as in A. n = 9 for each condition (i.e., n = 36 total), from three independent experiments, mean ± SEM; one-way ANOVA; multiple comparisons Tukey’s test was conducted to assess significance from control. p < 0.0001 denoted as ****.

Journal: bioRxiv

Article Title: A novel form of macropinocytosis mediates ultra-rapid transfer of pathological alpha-synuclein to lysosomes

doi: 10.1101/2022.01.06.475207

Figure Lengend Snippet: ( A ) Dopaminergic NPCs were incubated for 0, 2, 10, 30 min at 37°C following addition of Alex488-labelled PFF at 2 µg/mL. The cells were then washed with trypsin, fixed, and stained with lysotracker. Scale bar = 20 µm. ( B ) NPCs differentiated into dopaminergic neurons were processed as in A . Scale bar = 20 µm. ( C ) Human astrocytes were grown and mounted on coverslips. PFF was added to each coverslip at 1 µg/ml. Cells were incubated for 0, 2, 10, 30 min at 37°C following addition of Alex488-labelled PFF. Cells were washed with trypsin, fixed, permeabilized, and stained with LAMP1. Scale bar = 20 µm and 2.5 µm insets. ( D/E ) Colocalization of Lysotracker with PFF from experiments as in A and B. n = 6 for neurons and n = 6 for NPCs in each condition (i.e., n = 48 total), performed in three independent experiments. ( F ) Colocalization rate of LAMP1 with PFF from experiments as in A. n = 9 for each condition (i.e., n = 36 total), from three independent experiments, mean ± SEM; one-way ANOVA; multiple comparisons Tukey’s test was conducted to assess significance from control. p < 0.0001 denoted as ****.

Article Snippet: To further differentiate into dopaminergic neurons, neural progenitor medium was switched to dopaminergic neural differentiation medium (Brainphys Neuronal medium, STEMCELL Technologies 05790) supplemented with N2A Supplement A (STEMCELL Technologies; 07152), Neurocult SM1 Neuronal Supplement (STEMCELL Technologies; 05711), BDNF (20 ng/mL; Peprotech; 450-02), GDNF (20 ng/mL; Peprotech; 450-10), Compound E (0.1 μM; STEMCELL Technologies; 73954), db-cAMP (0.5 mM; Carbosynth; ND07996), Ascorbic acid (200 μM; Sigma; A5960) and laminin (1 μg/mL, Sigma; L2020).

Techniques: Incubation, Staining, Control

( A ) Dopaminergic neurons derived from human iPSCs mounted onto coverslips were given PFF488 at 2 µg/ml on ice for 1 h. Cells were removed from ice, given fresh media and were then incubated for 0 h, 1 day or 7 days at 37°C. Following incubation, cells were trypsin washed and fixed. Lysosomes were stained using LAMP1 (red) antibody. PFF remains localized to lysosomes 7 days following its addition to the cell. Scale bar = 20 µm for low magnification and 5 µm for insets. ( B ) Human astrocytes were mounted on coverslips and treated as described in A . PFF fluorescence builds in lysosomes over 7 days. Arrowheads point to LAMP1 and PFF colocalization. Scale bar = 20 µm for low magnification and 5 µm for insets.

Journal: bioRxiv

Article Title: A novel form of macropinocytosis mediates ultra-rapid transfer of pathological alpha-synuclein to lysosomes

doi: 10.1101/2022.01.06.475207

Figure Lengend Snippet: ( A ) Dopaminergic neurons derived from human iPSCs mounted onto coverslips were given PFF488 at 2 µg/ml on ice for 1 h. Cells were removed from ice, given fresh media and were then incubated for 0 h, 1 day or 7 days at 37°C. Following incubation, cells were trypsin washed and fixed. Lysosomes were stained using LAMP1 (red) antibody. PFF remains localized to lysosomes 7 days following its addition to the cell. Scale bar = 20 µm for low magnification and 5 µm for insets. ( B ) Human astrocytes were mounted on coverslips and treated as described in A . PFF fluorescence builds in lysosomes over 7 days. Arrowheads point to LAMP1 and PFF colocalization. Scale bar = 20 µm for low magnification and 5 µm for insets.

Article Snippet: To further differentiate into dopaminergic neurons, neural progenitor medium was switched to dopaminergic neural differentiation medium (Brainphys Neuronal medium, STEMCELL Technologies 05790) supplemented with N2A Supplement A (STEMCELL Technologies; 07152), Neurocult SM1 Neuronal Supplement (STEMCELL Technologies; 05711), BDNF (20 ng/mL; Peprotech; 450-02), GDNF (20 ng/mL; Peprotech; 450-10), Compound E (0.1 μM; STEMCELL Technologies; 73954), db-cAMP (0.5 mM; Carbosynth; ND07996), Ascorbic acid (200 μM; Sigma; A5960) and laminin (1 μg/mL, Sigma; L2020).

Techniques: Derivative Assay, Incubation, Staining, Fluorescence

(A) U2OS cells previously transfected with EBFP2-LAMP1 were transfected with CHC siRNA or control siRNA. Cell lysates were immunoblotted with antibodies recognizing the indicated proteins (B) At 24 h, control and CHC siRNA-treated cells were re-plated as a mosaic onto coverslips. PFF was added to each coverslip at 2 µg/ml. Cells were incubated for 0, 2, 10, 30 min at 37°C following addition of PFF. Cells were washed with trypsin and fixed. Arrowheads show large LAMP1- and PFF-positive vesicles in both CHC positive and CHC negative cells. KD cells are outlined with dashed lines. Scale bar = 20 µm. (C) Internalization of PFF in CHC KD vs control cells at 2 min were quantified from experiments as in B . n = 6 for each condition (i.e., n = 12 total), from three independent experiments, mean ± SEM; unpaired t-test; p > 0.05 denoted as ns for not significant. (D) Astrocytes were grown and mounted on coverslips. Cell media was replaced with serum-free media containing 20 µM EIPA, 5 µM LatB or DMSO (vehicle control) for 30 min. PFF (green) was added to each coverslip at 2 µg/ml for 0, 2, 10, 30 min at 37°C. Cells were trypsin washed and fixed. Cell nuclei were stained with DRAQ7. Scale bar = 20 µm. ( E/F ) Quantification of PFF and EGF uptake from experiments as in D . At each timepoint, control was compared to LatB and EIPA using unpaired samples t-test. n=8 for each condition (i.e., n = 48 total), from three independent experiments, mean ± SEM; one-way ANOVA done for neurons and NPC separately; multiple comparisons Tukey’s test was conducted to assess significance from control; p < 0.001 denoted as *** and p < 0.0001 denoted as ****. ( G ). Human dopaminergic NPCs were treated with LatA and PFF for 24 h before fixation. y-axis: % intracellular PFF-Alexa fluorescence relative to DMSO (vehicle) only control (DMSO=100%). X-axis: Latrunculin A treatment concentration in µM.

Journal: bioRxiv

Article Title: A novel form of macropinocytosis mediates ultra-rapid transfer of pathological alpha-synuclein to lysosomes

doi: 10.1101/2022.01.06.475207

Figure Lengend Snippet: (A) U2OS cells previously transfected with EBFP2-LAMP1 were transfected with CHC siRNA or control siRNA. Cell lysates were immunoblotted with antibodies recognizing the indicated proteins (B) At 24 h, control and CHC siRNA-treated cells were re-plated as a mosaic onto coverslips. PFF was added to each coverslip at 2 µg/ml. Cells were incubated for 0, 2, 10, 30 min at 37°C following addition of PFF. Cells were washed with trypsin and fixed. Arrowheads show large LAMP1- and PFF-positive vesicles in both CHC positive and CHC negative cells. KD cells are outlined with dashed lines. Scale bar = 20 µm. (C) Internalization of PFF in CHC KD vs control cells at 2 min were quantified from experiments as in B . n = 6 for each condition (i.e., n = 12 total), from three independent experiments, mean ± SEM; unpaired t-test; p > 0.05 denoted as ns for not significant. (D) Astrocytes were grown and mounted on coverslips. Cell media was replaced with serum-free media containing 20 µM EIPA, 5 µM LatB or DMSO (vehicle control) for 30 min. PFF (green) was added to each coverslip at 2 µg/ml for 0, 2, 10, 30 min at 37°C. Cells were trypsin washed and fixed. Cell nuclei were stained with DRAQ7. Scale bar = 20 µm. ( E/F ) Quantification of PFF and EGF uptake from experiments as in D . At each timepoint, control was compared to LatB and EIPA using unpaired samples t-test. n=8 for each condition (i.e., n = 48 total), from three independent experiments, mean ± SEM; one-way ANOVA done for neurons and NPC separately; multiple comparisons Tukey’s test was conducted to assess significance from control; p < 0.001 denoted as *** and p < 0.0001 denoted as ****. ( G ). Human dopaminergic NPCs were treated with LatA and PFF for 24 h before fixation. y-axis: % intracellular PFF-Alexa fluorescence relative to DMSO (vehicle) only control (DMSO=100%). X-axis: Latrunculin A treatment concentration in µM.

Article Snippet: To further differentiate into dopaminergic neurons, neural progenitor medium was switched to dopaminergic neural differentiation medium (Brainphys Neuronal medium, STEMCELL Technologies 05790) supplemented with N2A Supplement A (STEMCELL Technologies; 07152), Neurocult SM1 Neuronal Supplement (STEMCELL Technologies; 05711), BDNF (20 ng/mL; Peprotech; 450-02), GDNF (20 ng/mL; Peprotech; 450-10), Compound E (0.1 μM; STEMCELL Technologies; 73954), db-cAMP (0.5 mM; Carbosynth; ND07996), Ascorbic acid (200 μM; Sigma; A5960) and laminin (1 μg/mL, Sigma; L2020).

Techniques: Transfection, Control, Incubation, Staining, Fluorescence, Concentration Assay