NSCs were enzymatically dissociated and plated onto poly-d -lysine (0.1 mg/mL)- and laminin (10 μg/mL)-coated glass coverslips placed in 24-well plates at a density of 30,000 cells/well in proliferation medium. Subsequently, NSCs were left unlabeled or treated for 20 hours with 20 or 50 μg MIRB and washed to remove extraneous MIRB. To induce differentiation, growth factors were retrieved, and the NSCs were maintained in a medium consisting of Neurobasal-A with 1% glutamax, 2% B-27, 1% antibiotic–antimycotic, and 2% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA, USA). Differentiation into neuronal and glial cell types was assessed via immunocytochemistry after 10 days in culture. Five random fields per coverslip were assessed, and the percentage of cells (stained with the nuclear marker 4′,6-diamidino-2-phenylindole [DAPI]) expressing Tuj1 (for neurons), S100β (for astrocytes), or RIP (for oligodendrocytes) was counted under a ×20 lens. NSC lines (n=3) were grown in parallel and assessed in triplicate.
Antibiotic antimycotic
Manufactured by Atlanta Biologicals
Sourced in United States
Antibiotic/antimycotic is a laboratory product used to suppress the growth of bacteria, fungi, and other microorganisms in cell culture media and other laboratory applications. It contains a combination of antibiotics and antifungal agents to provide broad-spectrum protection against contamination.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Atlanta Biologicals (5 mentions)
Ebm 2 basal medium, by Lonza (3 mentions)
Chemically defined lipid concentrate, by Thermo Fisher Scientific (3 mentions)
Hepes, by Atlanta Biologicals (2 mentions)
Fibroblast growth factor (fgf), by Merck Group (2 mentions)
Facsaria 3, by BD (1 mentions)
Gentamycin, by Merck Group (1 mentions)
6 protocols using antibiotic antimycotic
1
Differentiation of Neural Stem Cells
2
Immortalized hCMEC/D3 Cell Culture for BBB
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The immortalized hCMEC/D3 cell line was donated by Dr. Couraud (INSERM, Paris). The hCMEC/D3 cells (passage 28–32) were seeded on collagen coated culture flasks (2.5-3 × 104/cm2) or glass slides (4 × 104/cm2) and maintained at 37°C with 5% CO2 exposure in EBM-2 basal medium (Lonza, Walkersville, MD, USA) supplemented with 5% FBS (Atlanta Biologicals, Lawrenceville, GA, USA), chemically defined lipid concentrate (Life Technologies, Carlsbad, CA, USA), growth factors, antibiotic/antimycotic (1:1, Atlanta Biologicals, GA, USA and HEPES (10 mM). Medium was changed every 2 days until the cells reached confluence. Monolayer integrity of hCMEC/D3 cells at confluence was confirmed by phase contrast microscopy and the expression of endothelial cell-specific phenotypic markers at cell-cell junctions, as previously described [26 (link)]. For treatment, cell monolayers were exposed to CSE concentration (5-20%) diluted from freshly prepared smoke extracts as described above. Cultures exposed to CSE-free vehicle (PBS) served as controls.
3
Cigarette Smoke Exposure on BBB Endothelium
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The hCMEC/D3 cells donated by Dr. Couraud (INSERM, Paris) (passage 29–30) were seeded on collagen coated cell culture flasks (2.0 × 104/cm2) and maintained at 37°C with 5% CO2 in EBM-2 basal medium (Lonza, Walkersville, MD, USA) supplemented with 5% FBS (Atlanta Biologicals, Lawrenceville, GA, USA), Fibroblast Growth Factor (Sigma Aldrich Inc), chemically defined lipid concentrate (Life Technologies, Carlsbad, CA, USA), antibiotic/antimycotic (1:1, Atlanta Biologicals, GA, USA and HEPES (10 mM). Medium was changed once after 2 days and then every other day until cells formed a continuous confluent monolayer. BBB endothelial monolayers were then chronically exposed (3 times/24 h) to a final 5% CSE concentration [8 (link)] derived from freshly prepared smoke extracts diluted in EBM-2 culture media. Following CSE exposure cells were processed for RNA and protein collection. Please note that a 5% CSE final concentration (using 3R4F cigarettes as reference) yields a nicotine output of ≈100 ng/mL, which is comparable to the steady state blood nicotine concentration measured in an average chronic smoker (≈20 cigarettes/day) [8 (link), 49 (link)–51 (link)]. Experiments related to nicotine exposure as a standalone agent where performed by directly diluting nicotine in the culture media at the desired concentration (100 ng/mL).
4
FIS1 Knockdown in HeLa Cells
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Hela cells were transiently transfected with FIS1 shRNA or Scramble shRNA. At 72 hours post-transfection, cells were trypsinized and sorted for GFP expression using a BD FACS Aria III cell sorter (Becton Dickinson) and collected in DMEM containing 10% FBS supplemented with 1X gentamycin (Millipore Sigma, St. Louis, MO) and 1X antibiotic-antimycotic (Atlanta Biologicals). GFP-positive cells were then seeded on coverslips for immunofluorescence studies and 96-well microplates for propidium iodide uptake assays. FIS1 knockdown was confirmed via immunoblotting using α-FIS1 as the primary antibody (Santa Cruz Biotechnology, Dallas, TX).
5
Differentiation Dynamics of Neural Stem Cells
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Previously established protocols were used to assess NSPC differentiation6 . NSPCs from each age group were enzymatically dissociated and plated on
poly-D-lysine and laminin-coated glass coverslips in 24 well plates. Growth factors were
retrieved to induce differentiation and cells were maintained in medium consisting of
Neurobasal-A with 1% GlutaMAX™, 2% B-27, 1% Antibiotic-Antimycotic, and 2%
fetal bovine serum (Atlanta Biologicals, Norcross, GA, USA). Immunocytochemical assessment
of differentiation into glial and neuronal cell types was performed after 10 days in
culture. Five fields per coverslip were enumerated and the percentage of DAPI-stained
cells expressing Tuj1 (neurons), glial fibrillary acidic protein (GFAP; astrocytes) or RIP
(oligodendrocytes) were counted under a 20× lens. A total of n = 3
independent NSPC lines, grown in parallel, were assessed in triplicate for the
analysis.
poly-D-lysine and laminin-coated glass coverslips in 24 well plates. Growth factors were
retrieved to induce differentiation and cells were maintained in medium consisting of
Neurobasal-A with 1% GlutaMAX™, 2% B-27, 1% Antibiotic-Antimycotic, and 2%
fetal bovine serum (Atlanta Biologicals, Norcross, GA, USA). Immunocytochemical assessment
of differentiation into glial and neuronal cell types was performed after 10 days in
culture. Five fields per coverslip were enumerated and the percentage of DAPI-stained
cells expressing Tuj1 (neurons), glial fibrillary acidic protein (GFAP; astrocytes) or RIP
(oligodendrocytes) were counted under a 20× lens. A total of n = 3
independent NSPC lines, grown in parallel, were assessed in triplicate for the
analysis.
6
hCMEC/D3 Endothelial Cell Culture
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The immortalized hCMEC/D3 endothelial cell line was obtained from Dr. Couraud (INSERM, Paris). These hCMEC/D3 cells (passages no. 28–30) were seeded on collagen-coated cell culture flasks or glass chamber slides (seeding density of 2.5 × 104/cm2) and maintained at 37°C with 5% CO2 exposure. Cell culture medium consisted of EBM-2 basal medium (Lonza, Walkersville, MD, USA), which was supplemented with 5% FBS (Atlanta Biologicals, Lawrenceville, GA, USA), Fibroblast Growth Factor (Sigma Aldrich), chemically defined lipid concentrate (Life Technologies, Carlsbad, CA, USA), antibiotic/antimycotic (1:1, Atlanta Biologicals, GA, USA) and HEPES (10 mM). The culture medium was changed every other day until the cells reached confluency. Phase contrast microscopy and the expression of characteristic phenotypic markers confirmed the monolayer integrity of the hCMEC/D3 cells at confluency.
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