Journal: Frontiers in Pharmacology

Article Title: A novel method for generating glutamatergic SH-SY5Y neuron-like cells utilizing B-27 supplement

doi: 10.3389/fphar.2022.943627

Figure Lengend Snippet: Methodology for testing B-27 for SH-SY5Y cell culture. (A) Initial B-27 tests. Before the start of the experiment, SH-SY5Y cells were cultured in DMEM/F-12 + 10% FBS in 10-cm diameter Petri dishes until they reached approximately 70% confluency. Once cells reached 70% confluency (day 0), 300,000 SH-SY5Y cells per well were seeded into 6-well plates in DMEM/F-12 + 10% FBS. By day 2, cells reached approximately 70% confluency and media was switched to either: DMEM/F-12 only, DMEM/F-12 + 10% FBS, DMEM/F-12 + 2% B-27 and DMEM/F-12 + 5% B-27. After 48 h (day 4), cells were imaged on the EVOS FLoid microscope ( Thermo Scientific ). (B) SH-SY5Y cells were cultured in the various media conditions for 48 h as described in part (A) . After 48 h (day 4), SH-SY5Y cells were either harvested in TRI reagent ( Zymo Research ) for RNA extraction, or the media was refreshed, and cells were cultured for a further 48 h. On day 6 (96-h exposure), SH-SY5Y cells were harvested in TRI reagent for RNA extraction. Following RNA extraction, RNA was converted into cDNA and qRT-PCR was performed on cells collected at both 48-h exposure and 96-h exposure time-points. DMEM/F-12, Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX supplement; FBS, fetal bovine serum; qRT-PCR, quantitative reverse transcription polymerase chain reaction. Figure made using BioRender.com .

Article Snippet: SH-SY5Y cells were cultured in 10-cm diameter Petri dishes ( Sigma Aldrich , #P7612-360EA) with 10 ml Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX (this is referred to simply as DMEM/F-12 in later sections) ( Fisher Scientific , #11524436) supplemented with 10% FBS ( Fisher Scientific , #11550356), and incubated at 37°C, 5% CO 2 , 95% humidity.

Techniques: Cell Culture, Microscopy, RNA Extraction, Quantitative RT-PCR, Modification, Reverse Transcription, Polymerase Chain Reaction

Journal: Frontiers in Pharmacology

Article Title: A novel method for generating glutamatergic SH-SY5Y neuron-like cells utilizing B-27 supplement

doi: 10.3389/fphar.2022.943627

Figure Lengend Snippet: B-27 encourages SH-SY5Y cells were seeded into 6-well plates at a density of 300,000 cells per well in DMEM/F-12 + 10% FBS. Once SH-SY5Y cells reached approximately 70% confluency, media was replaced with either: DMEM/F-12 only, DMEM/F-12 + 10% FBS, DMEM/F-12 + 2% B-27 supplement or DMEM/F-12 + 5% B-27 supplement. Cells were imaged using the EVOS FLoid microscope ( Thermo Scientific ) before media was replaced and 48 h after media was replaced. Scale bar = 100 μm. DMEM/F-12, Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX supplement; FBS, fetal bovine serum.

Article Snippet: SH-SY5Y cells were cultured in 10-cm diameter Petri dishes ( Sigma Aldrich , #P7612-360EA) with 10 ml Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX (this is referred to simply as DMEM/F-12 in later sections) ( Fisher Scientific , #11524436) supplemented with 10% FBS ( Fisher Scientific , #11550356), and incubated at 37°C, 5% CO 2 , 95% humidity.

Techniques: Microscopy, Modification

Journal: Frontiers in Pharmacology

Article Title: A novel method for generating glutamatergic SH-SY5Y neuron-like cells utilizing B-27 supplement

doi: 10.3389/fphar.2022.943627

Figure Lengend Snippet: SH-SY5Y cells cultured in B-27 show reduced growth rate compared to SH-SY5Y cells cultured in FBS. (A) On Day 0, 20,000 SH-SY5Y cells per well were seeded into 6-well plates with DMEM/F-12 + 10% FBS. On Day 1, media was replaced with either DMEM/F-12 + 10% FBS or DMEM/F-12 + 2% B-27 supplement, and the number of cells per well was counted using a hemocytometer. Cell counts were performed at the same time daily for 9 days. (B) Representative brightfield mages were taken on Day 1, Day 5, and Day 9 using the EVOS FLoid microscope ( Thermo Scientific ). Scale bar = 100 μm. (C) On Day 0, 5,000 SH-SY5Y cells per well were seeded into 24-well plates with DMEM/F-12 + 10% FBS. On Day 1, media was replaced with either DMEM/F-12 + 10% FBS or DMEM/F-12 + 2% B-27 supplement. Cells were fixed on Day 1, Day 3, Day 5 and Day 8 with 4% PFA for 20 min. Proliferating cells were stained with 1:400 rabbit anti-Ki67 and visualized with 1:400 Alexa Fluor 555-conjugated donkey anti-rabbit ( Invitrogen , #A31572) (red). Nuclei were counterstained with 1:5,000 DAPI (blue). Images taken at ×20 magnification through the DAPI (excitation 325–375 nm; emission 435–485 nm) and the TXR (excitation 540–580 nm; emission 592–668 nm) filter cubes on the Leica DMi8 Widefield microscope. Scale bar = 100 μm. (D) The number of Ki67+ cells and DAPI-stained nuclei were counted using the FIJI software, and the percentage of Ki-67+ cells per image calculated. Three independent replicates were performed, and 15 random images were analyzed per condition. Data presented as mean ± SEM. Statistical significance against the DMEM/F-12 + 10% FBS condition was determined using Unpaired t tests in GraphPad Prism version 9.0.0 for Mac, GraphPad Software, San Diego, California, United States, www.graphpad.com . ns = not significant; * p < 0.05; **** p < 1 × 10 –4 . DAPI: 4′,6-Diamidino-2-phenylindole; DMEM/F-12, Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX supplement; FBS, Fetal bovine serum; PFA, Paraformaldehyde; SEM, standard error of the mean.

Article Snippet: SH-SY5Y cells were cultured in 10-cm diameter Petri dishes ( Sigma Aldrich , #P7612-360EA) with 10 ml Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX (this is referred to simply as DMEM/F-12 in later sections) ( Fisher Scientific , #11524436) supplemented with 10% FBS ( Fisher Scientific , #11550356), and incubated at 37°C, 5% CO 2 , 95% humidity.

Techniques: Cell Culture, Microscopy, Staining, Software, Modification

Journal: Frontiers in Pharmacology

Article Title: A novel method for generating glutamatergic SH-SY5Y neuron-like cells utilizing B-27 supplement

doi: 10.3389/fphar.2022.943627

Figure Lengend Snippet: Longer term B-27 exposure leads to SH-SY5Y cell cycle arrest. SH-SY5Y cells were cultured in DMEM/F-12 only, or DMEM/F-12 supplemented with either 10% FBS, 2% B-27 or 5% B-27 for (A) 48 h or (B) 96 h. qRT-PCR was performed to measure mRNA expression of cell cycle markers CDK6 and CDK1 , and apoptosis marker CASP3 . Relative expression of CDK1 , CDK6 , and CASP3 (standardized to GAPDH and POLR2A ) normalized to the DMEM/F-12 + 10% FBS culture conditions. Data presented as mean ± SEM. N = 3 independent biological samples with two to three technical replicates each. Statistical significance against the DMEM/F-12 + 10% FBS condition was determined using ordinary one-way ANOVA and Tukey’s multiple comparison tests in GraphPad Prism version 9.0.0 for Mac, GraphPad Software, San Diego, California, United States, www.graphpad.com . ns = not significant; * p < 0.05; ** p < 0.01; **** p < 1 × 10 –4 . CASP3, Caspase 3 ; CDK1, Cyclin dependent kinase 1; CDK6, Cyclin dependent kinase 6; DMEM/F-12, Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX supplement; FBS, fetal bovine serum; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase ; mRNA, messenger RNA; POLR2A , RNA polymerase II subunit A ; SEM, standard error of the mean.

Article Snippet: SH-SY5Y cells were cultured in 10-cm diameter Petri dishes ( Sigma Aldrich , #P7612-360EA) with 10 ml Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX (this is referred to simply as DMEM/F-12 in later sections) ( Fisher Scientific , #11524436) supplemented with 10% FBS ( Fisher Scientific , #11550356), and incubated at 37°C, 5% CO 2 , 95% humidity.

Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Marker, Comparison, Software, Modification

Journal: Frontiers in Pharmacology

Article Title: A novel method for generating glutamatergic SH-SY5Y neuron-like cells utilizing B-27 supplement

doi: 10.3389/fphar.2022.943627

Figure Lengend Snippet: B-27 encourages neurite outgrowth and differentiation in SH-SY5Y cells. SH-SY5Y cells were cultured in DMEM/F-12 supplemented with either 10% FBS, 2% B-27 supplement, 5% B-27 supplement or 0.5% FBS + 20 ng/ml BDNF + 10 μM RA for 5 days. Cells were fixed with 4% PFA for 20 min. Microtubules were stained with 1:500 mouse anti-β(III)-Tubulin ( R&D Systems , #MAB1195) and visualized with 1:400 Alexa Fluor ® 488-conjugated donkey anti-mouse ( Thermo Scientific , #A21202) (green). Nuclei were counterstained with 1:5,000 DAPI (blue). Images taken at ×20 magnification through the DAPI channel (excitation 325–375 nm; emission 435–485 nm) and the FITC channel (excitation 460–500 nm; emission 512–542 nm) on the Leica DM4000 LED upright fluorescent microscope. Scale bar = 100 μm. BDNF, brain-derived neurotrophic factor; DAPI, 4′,6-Diamidino-2-phenylindole; DMEM/F-12, Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX supplement; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; PFA, Paraformaldehyde; RA, retinoic acid.

Article Snippet: SH-SY5Y cells were cultured in 10-cm diameter Petri dishes ( Sigma Aldrich , #P7612-360EA) with 10 ml Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX (this is referred to simply as DMEM/F-12 in later sections) ( Fisher Scientific , #11524436) supplemented with 10% FBS ( Fisher Scientific , #11550356), and incubated at 37°C, 5% CO 2 , 95% humidity.

Techniques: Cell Culture, Staining, Microscopy, Derivative Assay, Modification

Journal: Frontiers in Pharmacology

Article Title: A novel method for generating glutamatergic SH-SY5Y neuron-like cells utilizing B-27 supplement

doi: 10.3389/fphar.2022.943627

Figure Lengend Snippet: B-27 induces SH-SY5Y cell differentiation. SH-SY5Y cells were cultured in DMEM/F-12 only, or DMEM/F-12 supplemented with either 10% FBS, 2% B-27 or 5% B-27 for (A) 48 h or (B) 96 h. qRT-PCR was performed to measure mRNA expression of differentiation markers, GAP43 , TUBB3 and SYP . Relative expression of GAP43 , TUBB3 , and SYP (standardized to GAPDH and POLR2A ) normalized to the DMEM/F-12 + 10% FBS culture conditions. Data presented as mean ± SEM. N = 3 independent biological samples with two to three technical replicates each. Statistical significance against the DMEM/F-12 + 10% FBS condition was determined using ordinary one-way ANOVA and Tukey’s multiple comparison tests in GraphPad Prism version 9.0.0 for Mac, GraphPad Software, San Diego, California, United States, www.graphpad.com . ns = not significant; * p < 0.05; ** p < 0.01; *** p < 1 × 10 –3 ; **** p < 1 × 10 –4 . DMEM/F-12, Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX supplement; FBS, fetal bovine serum; GAP43 , Growth associated protein 43 ; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase ; mRNA, messenger RNA; POLR2A , RNA polymerase II subunit A ; SEM, standard error of the mean; SYP , Synaptophysin ; TUBB3, Tubulin beta 3 class III .

Article Snippet: SH-SY5Y cells were cultured in 10-cm diameter Petri dishes ( Sigma Aldrich , #P7612-360EA) with 10 ml Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX (this is referred to simply as DMEM/F-12 in later sections) ( Fisher Scientific , #11524436) supplemented with 10% FBS ( Fisher Scientific , #11550356), and incubated at 37°C, 5% CO 2 , 95% humidity.

Techniques: Cell Differentiation, Cell Culture, Quantitative RT-PCR, Expressing, Comparison, Software, Modification

Journal: Frontiers in Pharmacology

Article Title: A novel method for generating glutamatergic SH-SY5Y neuron-like cells utilizing B-27 supplement

doi: 10.3389/fphar.2022.943627

Figure Lengend Snippet: SH-SY5Y cells differentiated with B-27 show no significant difference in neurite length compared with SH-SY5Y cells differentiated with BDNF. (A) SH-SY5Y cells were differentiated for 5 days with DMEM/F-12 + 10 μM RA, in conjunction with varying combinations of neurite outgrowth-stimulating molecules (B-27 or BDNF) or serum starvation (0.5% FBS). Cells were fixed with 4% PFA for 20 min. Microtubules were stained with 1:500 mouse anti-β(III)-Tubulin ( R&D Systems , #MAB1195) and visualized with 1:400 Alexa Fluor ® 488-conjugated donkey anti-mouse ( Thermo Scientific , #A21202) (green). Nuclei were counterstained with 1:5,000 DAPI (blue). Images taken at ×20 magnification. Scale bar = 100 μm. (B) Longest neurite length and total neurite length measurements were taken using the FIJI software . Three independent experimental replicates were performed and 80 cells per condition were measured in total. Data presented as mean ± SEM. Statistical significance against the DMEM/F-12 + 10% FBS condition was determined using ordinary one-way ANOVA and Tukey’s multiple comparison tests in GraphPad Prism version 9.0.0 for Mac, GraphPad Software, San Diego, California, United States, www.graphpad.com . ns = not significant. BDNF, brain-derived neurotrophic factor; DAPI, 4′,6-Diamidino-2-phenylindole; DMEM/F-12, Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX supplement; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; PFA, Paraformaldehyde; RA, retinoic acid.

Article Snippet: SH-SY5Y cells were cultured in 10-cm diameter Petri dishes ( Sigma Aldrich , #P7612-360EA) with 10 ml Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX (this is referred to simply as DMEM/F-12 in later sections) ( Fisher Scientific , #11524436) supplemented with 10% FBS ( Fisher Scientific , #11550356), and incubated at 37°C, 5% CO 2 , 95% humidity.

Techniques: Staining, Software, Comparison, Derivative Assay, Modification

Journal: Frontiers in Pharmacology

Article Title: A novel method for generating glutamatergic SH-SY5Y neuron-like cells utilizing B-27 supplement

doi: 10.3389/fphar.2022.943627

Figure Lengend Snippet: SH-SY5Y cells differentiate into a more glutamatergic-like phenotype following treatment with B-27. (A) SH-SY5Y cells were cultured in DMEM/F-12 supplemented with either 10% FBS, 2% B-27 or 5% B-27 for 96 h. qRT-PCR was performed to measure mRNA expression of cholinergic marker SLC18A1 (VMAT1), dopaminergic marker TH and glutamatergic markers GLUL , SLC17A7 (VGLUT1) and GLS . Relative expression of SLC18A1, TH, GLUL, SLC17A7 and GLS (standardized to GAPDH and POLR2A ) normalized to the DMEM/F-12 + 10% FBS culture conditions. Data presented as mean ± SEM; N = 3 independent biological samples with two to three technical replicates each. Statistical significance was determined using ordinary one-way ANOVA and Tukey’s multiple comparison tests in GraphPad Prism version 9.0.0 for Mac, GraphPad Software, San Diego, California, United States, www.graphpad.com . ns = not significant; * p < 0.05; ** p < 0.01; *** p < 1 × 10 –3 ; **** p < 1 × 10 –4 . (B) Western blot of lysate from SH-SY5Y cells cultured in DMEM/F-12 supplemented with either 10% FBS or 5% B-27 for 96 h. Membranes were probed with rabbit anti-GLS ( Proteintech #29519-1-AP), mouse anti-TH ( Proteintech , #66334-1-Ig), mouse anti-GLUL ( Proteintech , #66323-1-Ig) and mouse anti-GAPDH ( Santa Cruz Biotechnology , #sc-47724). GAPDH was used as a loading control. Molecular weights are as follows GLS = 58 kDa and 65 kDa; TH = 55 kDa; GLUL = 42 kDa; GAPDH = 36 kDa. (C) Levels of protein expression relative to GAPDH were quantify through densitometric analysis using the FIJI software . Three independent experiments were performed and densitometric analysis performed using seven independent blots. Data presented as mean ± SEM. Statistical significance against the DMEM/F-12 + 10% FBS condition was determined using Unpaired t tests in GraphPad Prism version 9.0.0 for Mac, GraphPad Software, San Diego, California, United States, www.graphpad.com . **** p < 1 × 10 –4 . DMEM/F-12, Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX supplement; FBS, fetal bovine serum; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase ; GLS, Glutaminase ; GLUL, Glutamate-ammonia ligase ; mRNA: messenger RNA; POLR2A, RNA polymerase II subunit A ; SEM, standard error of the mean; SLC17A7, Solute carrier family 17 member 7 ; SLC18A1, Solute carrier family 18 member A1; TH, tyrosine hydroxylase ; VGLUT1, vesicular glutamate transporter 1 ; VMAT1, Vesicular monoamine transporter 1 .

Article Snippet: SH-SY5Y cells were cultured in 10-cm diameter Petri dishes ( Sigma Aldrich , #P7612-360EA) with 10 ml Dulbecco’s modified eagle medium/nutrient mixture F-12 with GlutaMAX (this is referred to simply as DMEM/F-12 in later sections) ( Fisher Scientific , #11524436) supplemented with 10% FBS ( Fisher Scientific , #11550356), and incubated at 37°C, 5% CO 2 , 95% humidity.

Techniques: Cell Culture, Quantitative RT-PCR, Expressing, Marker, Comparison, Software, Western Blot, Control, Modification