From: A synthetic pregnenolone analog promotes microtubule dynamics and neural development
Antibodies | Source | Identifier | Note |
---|---|---|---|
Mouse monoclonal anti-acetylated tubulin, clone 6-11B-1 | Sigma | Cat#T6793 | 1:500 |
Rabbit monoclonal anti-III-beta tubulin [EP1569Y] | Abcam | Cat#ab52623 | 1:500 |
Rabbit polyclonal anti-beta tubulin | Proteintech | 10094-1-AP | 1:200 |
Goat anti-mouse IgG (H+L) superclonal recombinant secondary antibody, Alexa Fluor 555 | Invitrogen | A28180 | 1:1000 |
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Oregon Green 488 | Invitrogen | O-11038 | 1:4000 |
Experimental models | Source | Identifier | Note |
---|---|---|---|
Mouse: C57BL/6J strain | National Applied Research Laboratories | Â | Â |
Zebrafish: TL strain | Â | Â | Â |
COS1 cells | Â | Â | Â |
Plasmids | Source | Identifier | Note |
---|---|---|---|
pSC2 + mOrange2-Flag-CLIP1 | This paper | N/A | Fluorescent CLIP1 for comet tracking |
Steroids | Source | Identifier | Note |
---|---|---|---|
Pregnenolone, 5-pregnen-3β-ol-20-one | Sigma-Aldrich | P9129 |  |
P5 derivatives | This paper | N/A | Â |
Reagents for cell culture | Source | Identifier | Note |
---|---|---|---|
Dulbecco’s Modified Eagle Medium | Gibco | 12100-046 | COS1 culture |
HyClone Characterized Fetal Bovine Serum | GE Life Sciences | SH30396.03 | COS1 culture |
Neurobasal Plus Medium | Gibco | A35829-01 | Primary neuronal culture |
Glutamax-I (100X) | Gibco | 35050-061 | Primary neuronal culture |
B27 Supplement (50X) | Gibco | 17504044 | Primary neuronal culture |
Penicillin–Streptomycin, liquid | Gibco | 15140-122 | Primary neuronal culture |
HEPES, 1Â M buffer solution | Gibco | 15630-080 | Primary neuronal culture |
HBSS, 10X | Gibco | 14185-052 | Primary neuronal culture |
Percoll pH 8.5–9.5 | Sigma-Aldrich | P1644 | Primary neuronal culture |
Poly-L-ornithine, 0.01% | Sigma-Aldrich | P4957 | Primary neuronal culture |
Trypsin–EDTA | Gibco | 25300-054 | Primary neuronal culture |
Trypsin inhibitor from Glycine max (soybean) | Sigma-Aldrich | T6522 | Primary neuronal culture |
DNase I | Roche | 04716728001 | Primary neuron harvest |
In vitro microtubule polymerization assay | Source | Identifier | Note |
---|---|---|---|
Tubulin protein (> 99% pure): porcine brain | Cytoskeleton | T240 |  |
Tubulin protein (rhodamine): porcine brain | Cytoskeleton | TL590M | Â |
GpCpp | Jena Biosciences | NU-405 | Â |
Paclitaxel from Taxus brevifolia, ≥ 95% (HPLC), powder | Sigma Aldrich | T7402 |  |
0.5 mL, Open-Top Thickwall Polycarbonate Tube, 8 × 34 mm | Beckman Coulter | 343776 |  |
Device | Source | Identifier | Note |
---|---|---|---|
Neon Transfection System | Invitrogen | MPK5000 | Â |
DeltaVision Core | GE Healthcare | N/A | Live cell imaging for comet tracking |
Zeiss LSM710 confocal microscope | Carl Zeiss | N/A | Neurons imaging |
Software | Source | Note | Â |
---|---|---|---|
ImarisFileConverter × 64 9.7.2 | Oxford Instruments |  |  |
Imaris × 64 9.7.2 | Oxford Instruments | Quantifying cerebellum volume |  |
SoftMax Pro 5.3 | Molecular Devices | Â | Â |
EnSpire | PerkinElmer | Â | Â |
ImageJ/Fiji | Base program for MTrackJ | Â | |
softWoRx | Applied Precision | Â | Â |
MTrackJ | [47] | CLIP1 comet quantification | Â |
ZEN 2.6 | Carl Zeiss | Image processing | Â |
GraphPad Prism 8.0.0 | Statistical analysis | Â |
Reagents for transfection and staining | Source | Identifier | Note |
---|---|---|---|
Neon Transfection System 100 µL Kit | Invitrogen | MPK10096 | pSC2+mOrange2-Flag-CLIP1 transfection |
DAPI | Life Technologies | D1306 | 1:10000 |
Rhodamine phalloidin | Cytoskeleton | PHDR1 | Â |
ProLong Gold Antifade Reagent | Invitrogen | P36930 | Â |