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Method Article

Cellular Models for Studying Alexander Disease: Functional Analysis of Primary Rat Astrocytes

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DOI:

10.3791/72930

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September 8th, 2026

 , 
1Institute of Molecular Medicine, College of Life Sciences and Medicine, National Tsing Hua University, 2School of Medicine, College of Life Sciences and Medicine, National Tsing Hua University

In This Article

Summary

We develop a primary rat astrocyte model that provides a physiologically relevant system for studying Alexander disease (AxD), enabling investigation of GFAP aggregation, pathological modification, and astrocyte dysfunction. This system bridges in vitro and in vivo studies, offering a robust platform to explore AxD pathology and identify therapeutic targets for neurodegenerative diseases.

Abstract

Astrocytes play a vital role in maintaining central nervous system homeostasis, supporting neuronal function, and responding to injury or disease. Dysfunction in astrocyte activity is increasingly recognized as a key contributor to neurodegenerative disorders, including Alexander disease (AxD), a rare and fatal condition caused by mutations in glial fibrillary acidic protein (GFAP). These mutations result in GFAP aggregation, Rosenthal fiber formation, and progressive astrocyte dysfunction. While in vivo animal models offer a complex cellular environment, both current in vitro and in vivo models face limitations in accurately replicating the intricate cellular context and functions of astrocytes. To address this, we developed a cellular model using primary rat astrocytes derived from wild-type (WT) and AxD model rats to study the functional effects of GFAP mutations. This physiologically relevant system enables detailed investigation of astrocyte-specific mechanisms, including GFAP aggregation, oxidative stress responses, and pathological modifications. For researchers without access to AxD model rats, lentiviral transduction offers an alternative method to introduce AxD-associated GFAP mutations into astrocytes derived from normal rats, thereby broadening the applicability of this approach. Compared with immortalized astrocyte cell lines or in vitro studies using recombinant protein, primary astrocytes better preserve native cellular architecture and molecular profiles, offering a robust platform for studying GFAP dynamics, solubility, and astrocytic responses to stressors such as oxidative damage and pathological modifications. This cellular model bridges the gap between molecular and systemic studies, providing a controlled experimental framework to explore astrocyte dysfunction in AxD. By complementing existing methodologies, primary astrocyte cultures enhance our understanding of AxD pathology and represent a valuable tool for identifying potential therapeutic targets for neurodegenerative diseases.

Introduction

Astrocytes are specialized glial cells that span the entire central nervous system (CNS), playing critical roles in maintaining brain homeostasis, regulating neuronal activity, and responding to injury or disease1. Their functions include neurotransmitter clearance and recycling, extracellular ion and water homeostasis, and support for neural circuit function. Astrocyte-secreted molecules are essential for synapse development and synaptic plasticity in the adult brain2. In response to CNS injury or disease, astrocytes undergo reactive changes that can mediate either pro- or anti-inflammatory signaling, enabling neuroprotective functions but potentially contributing to CNS pathologies depending on the context3. Dysregulation of astrocyte function is increasingly recognized as a key factor in CNS health and disease4,5.

Glial fibrillary acidic protein (GFAP), a type III intermediate filament (IF) protein, is a major cytoskeletal component of astrocytes, expressed by most but not all astrocytes. GFAP is upregulated in response to CNS injury, serving as a marker for tissue damage severity6. Although GFAP is traditionally associated with structural roles7, its functional implications remain underexplored. Mutations in GFAP cause Alexander disease (AxD)8, a rare neurodegenerative disorder characterized by GFAP aggregation, Rosenthal fiber formation, and progressive astrocyte dysfunction. While in vitro biochemical studies9,10,11 and in vivo animal models12,13 have provided insights into AxD pathology, they fail to capture the complex cellular context of astrocyte function14.

To overcome these limitations, we developed a cellular model using primary rat astrocytes derived from wild-type (WT) and AxD model rats13. This system preserves the native cellular architecture and molecular profiles of astrocytes, enabling the study of GFAP aggregation, oxidative stress responses, and pathological modifications associated with AxD. For researchers without access to AxD model rats, lentiviral transduction offers an alternative method to introduce AxD-associated GFAP mutations into astrocytes derived from normal rats10.

Compared with immortalized cell lines, primary astrocytes preserve the heterogeneity and complexity of astrocyte populations in vivo, providing a physiologically relevant platform for investigating astrocyte-specific mechanisms15. Unlike in vitro studies using recombinant protein9, primary astrocytes naturally express GFAP and other astrocyte-specific markers, allowing for the study of disease-associated variants within a native cellular environment10,16,17. Furthermore, primary astrocytes facilitate investigation into functional responses to stress stimuli, such as oxidative damage18 and inflammation19, which are challenging to replicate in simplified in vitro systems.

In AxD research, primary astrocytes complement biochemical and in vivo approaches by bridging the gap between molecular and systemic studies. While biochemical assays focus on the molecular properties of GFAP mutations, they lack cellular complexity, whereas in vivo models capture systemic effects but are less suited for mechanistic cellular studies. Primary astrocytes provide an intermediate system with cellular complexity and experimental control, aligning with current trends in neurodegenerative disease research that emphasize cellular models for understanding disease mechanisms and identifying therapeutic targets20.

This manuscript presents the development and application of primary rat astrocytes as a cellular model for AxD research. By enabling direct comparison between WT and AxD astrocytes, this approach addresses key limitations of alternative techniques and expands accessibility via lentiviral transduction. These findings contribute to a deeper understanding of astrocyte dysfunction in AxD and offer a valuable tool for advancing therapeutic discoveries in neurodegenerative diseases.

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Protocol

Animals used in this protocol were approved by the Institutional Animal Care and Use Committee of the College of Life Sciences and Medicine at the National Tsing Hua University (NTHU IACUC Approval No. 109088 and 111060) and in accordance with the guidelines of the Agriculture Guidebook for the Care and Use of Laboratory Animals.

1. Preparation of media, coverslips, dishes, and plates 

  1. Before starting, ensure the following solutions (Table 1) are prepared and ready for use.
  2. Sterilize scissors and forceps by autoclaving before use to ensure sterility.
  3. Prepare poly-L-lysine (PLL) solution at 0.02 mg/mL in boric acid buffer.
  4. Add enough PLL solution to fully cover the surface of the coverslip or the bottom of the culture dish/plate. Ensure the coating is even.
  5. Leave the PLL-coated coverslips and dishes to incubate overnight at room temperature in a laminar flow hood.  
  6. The next day, carefully remove the PLL solution. Rinse the coated coverslips or dish/plate three times with fresh sterile water to remove any residual PLL.
    NOTE: It is critical to thoroughly rinse the PLL-coated flask/plate, or the coverslips, to remove residual PLL that may negatively affect cell viability. 

2. Preparation of primary astrocyte culture

NOTE: A schematic overview is shown in Figure 1.

  1. Following the induction of hypothermia by placing the pups on ice, decapitate the postnatal day 1–3 rat pup and separate the head from the body.
  2. Place the head in a 60-mm dish and secure the sides with forceps. Using fine scissors, gently dissect the skin on the top of the head.
  3. Make an incision at the vertex of the head using fine scissors to carefully open the skull. Gently separate and remove the two halves of the skull.
  4. Using forceps, pinch off the brain from the base and transfer it to a 60-mm dish containing dissection medium.
  5. Place each hemisphere with the medial surface facing upward to expose the midline structures.
  6. Under a dissecting microscope, gently remove the midbrain and brainstem tissues, leaving intact hemispheres containing the cortex and hippocampus.
  7. Use fine forceps to gently peel off the meninges, ideally as a single piece. Carefully check for any remaining meninges hidden in fissures and remove them completely.
  8. Move the dissected brain tissue to a laminar flow hood. Aspirate the dissection medium using a 1 mL pipette and add fresh dissection medium.
  9. Finely chop the tissue using a sterile scalpel blade.
  10. Transfer the chopped tissue into 5 mL of dissociation medium in a 15 mL tube. Incubate the tube in a 37ºC water bath for 15 min, gently shaking the tube every 5 min to ensure proper enzyme-tissue interaction.
  11. After incubation, allow the tissue to settle at the bottom of the tube. Aspirate the supernatant and add 5 mL of dissection medium.
  12. Invert the tube 5 times to wash the tissue. Let the tube stand for 3 min to allow the tissue to settle, then repeat the washing process three times using fresh dissection medium.
  13. After the final wash, remove the supernatant and add 2.5 mL of dissection medium and 2.5 mL of maintenance medium.
  14. Triturate the tissue by passing the solution up and down 10–15 times using a 10-mL pipette.
  15. Let the tube stand for 3 min to allow the tissue to settle. Transfer the supernatant (containing cells) to a new 50 mL conical tube.
  16. Add 5 mL of MEM medium to the original tube containing the settled tissue. Triturate another 10–15 times with a 10-mL pipette until most chunks disappear.
  17. Pass the cell suspension through a sterile 40 µm cell strainer into a clean 50 mL conical tube containing 5 mL of maintenance medium.
  18. Centrifuge the filtered cell suspension at 200 × g for 5 min at room temperature.
  19. Remove the supernatant, then gently loosen the cell pellet by tapping the tube. 
  20. Add 5 mL of MEM medium and pipette up and down 10 times to resuspend the pellet. Keep the cell suspension in a 37ºC water bath during cell counting.
  21. Dilute 10 μL of the cell suspension with 90 µL of MEM medium. Mix well using a 200 µL pipette tip.
  22. Add 20 μL of diluted cell suspension to 20 µL of trypan blue. Count the cells using a hemocytometer.
  23. Plate cells in culture vessels as follows: for a 24-well plate, seed 2 × 10⁵ cells per well; for a 6-well plate, seed 5 × 10⁵ cells per well; for a 6-cm dish, seed 1 × 10⁶ cells per dish.
  24. Place the plates or dishes in a tissue culture incubator. Avoid leaving the cells at room temperature for extended periods.
  25. Examine the cells under a microscope to ensure they have adhered to the substrate 4 h after plating.
  26. At 24 h after plating, replace the culture medium with fresh maintenance medium.
  27. Feed the culture every 2–3 days by replacing half of the medium with fresh maintenance medium.
  28. After 7 days, the cells should reach approximately 80% confluency and will be ready for experiments.
    NOTE: To ensure optimal conditions for astrocyte cultures, perform all steps in a sterile environment, preferably within a laminar flow hood, to avoid contamination. Additionally, ensure that all reagents are pre-warmed to 37°C where applicable. Be meticulous when removing meninges, as any leftover meningeal tissue can interfere with the cultures. Furthermore, avoid leaving cells at room temperature for extended periods during plating and counting to maintain cell viability and integrity.

3. Production of lentiviral particles and transduction 

NOTE: A schematic overview is shown in Figure 2.

  1. Seed HEK293T cells in a 10 cm2 petri dish containing fresh culture medium (Table 1).
  2. Grow cells overnight at a 37ºC incubator to reach 50%–60% confluency.
  3. In 1 mL of Opti-MEM, add 20 µg of lentiviral plasmid encoding GFAP or its variants, 5.6 µg of packaging vector, and 1.8 µg of envelope vector. 
  4. Add 45 µL of transfection reagent to the DNA-containing solution.
  5. Mix gently and incubate at room temperature for 15 min.
  6. Add the transfection solution directly to the HEK293T cells and incubate at 37°C cell culture incubator for 4 h.
  7. Aspirate the transfection solution and replace it with fresh culture medium.  
  8. Continue culturing the cells for 2 days.
  9. Collect the cell culture supernatant and replace it with fresh culture medium.
  10. The next day, collect the cell culture supernatant again and replace it with fresh culture medium.
  11. The following day, collect the final cell culture supernatant.
  12. Combine the supernatants from steps 3.9–3.11 into a 50-mL conical tube.
  13. Filter the combined supernatant through a 0.45 µm filter using a 60 mL syringe to remove debris.  
  14. Centrifuge the filtered supernatant at 1,400 × g for 5 min at room temperature to remove dead cells.
  15. Transfer the cleared supernatant to ultracentrifuge tubes.
  16. Centrifuge at 100,000 × g for 70 min at 4°C to pellet the lentiviral particles.
  17. Carefully remove the supernatant without disturbing the pellet at the bottom of the tube.
  18. Resuspend the pellet in 600 µL of Opti-MEM medium by gently pipetting up and down.
  19. Divide the resuspended lentiviral particles into 50 μL aliquots and store them at -80°C until needed. 
  20. Seed primary astrocytes in a 6-well plate and allow them to reach the 50%–70% confluency (Section 2).
  21. Thaw the required amounts of lentiviral particles on ice.
  22. Add 50 µL of lentiviral particles to each well, supplemented with 8 µg/mL Polybrene.
  23. Gently swirl the plate to ensure even distribution of the virus and Polybrene. Incubate the cells in a 37ºC cell culture incubator for 4–8 h.
  24. Aspirate the virus-containing medium carefully to avoid disturbing the cells.
  25. Replace the medium with fresh culture medium and continual culture for 2–3 days.
  26. Perform downstream analyses on the transduced cells using immunofluorescence microscopy to visualize intermediate filament network formation, or immunoblotting to assess GFAP expression.
    NOTE: When handling lentiviral particles, it is crucial to use appropriate personal protective equipment (PPE) to ensure safety. Additionally, to maintain the integrity of the viral particles, avoid repeated freeze-thaw cycles.

4. Immunofluorescence microscopy 

  1. Gently aspirate the culture medium from the wells containing coverslips with plated cells. Avoid disturbing the cells.
  2. Fix cells by adding one of the following fixative solutions: For primary astrocytes, use 3.2% (wt/vol) paraformaldehyde; for astrocyte-neuron culture, use 3.2% (wt/vol) paraformaldehyde and 4% (wt/vol) sucrose.
  3. Incubate the coverslips in the fixative for 15 min at room temperature. Unless otherwise noted, perform all subsequent steps at room temperature. 
  4. Rinse the fixed cells three times with phosphate-buffered saline (PBS) to remove the fixative.
  5. Permeabilize the cells by incubating them in 0.2% (vol/vol) Triton X-100 prepared in blocking buffer (5% (vol/vol) normal goat serum in PBS) for 15 min. 
  6. Rinse the permeabilized cells three times with PBS.
  7. Prepare the primary antibody solution by diluting the antibody in blocking buffer according to the manufacturer's recommended concentration.
  8. Incubate the coverslips in the primary antibody solution for at least 1 h at room temperature.
  9. Rinse the coverslips three times with PBS to remove unbound primary antibody.
  10. Prepare the secondary antibody solution by diluting goat anti-mouse and goat anti-rabbit secondary antibodies conjugated fluorophores in blocking buffer at a 1:500 dilution.  
  11. Incubate the coverslips in the secondary antibody solution for 1 h at room temperature.
  12. Rinse the coverslips three times with PBS to remove unbound secondary antibody.
  13. Carefully transfer the coverslips onto glass slides using tweezers.
  14. Mount the coverslips with mounting medium to preserve fluorescence.
  15. Gently seal the edges of the coverslip with clear nail polish to prevent movement and evaporation.
  16. Allow the nail polish to dry completely before proceeding to imaging.
  17. The prepared slides are ready for imaging using an immunofluorescence or confocal microscope.
    NOTE: To ensure the success of experiments, prepare all reagents, such as PBS, blocking buffer, and antibodies, fresh and maintain sterility. Handle coverslips with care to avoid damaging the cells or scratching the glass surface. Protect samples from light during antibody incubation and after mounting to prevent photobleaching of fluorescent signals. Additionally, optimize antibody concentrations and incubation times according to the specific experimental setup.

5. Immunoblotting

  1. Rinse the astrocytes twice with PBS to remove residual medium and debris.
  2. Place the plate or dish on ice to minimize protein degradation. Working one well at a time, completely aspirate the PBS.
  3. Add ice-cold radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors. For a 6-well plate, add 250 µL per well; for a 6-cm dish, add 500 µL per dish.
  4. Use a cell scraper to thoroughly scrape all astrocytes into the RIPA buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% (wt/vol) NP-40, 5 mM ethylenediaminetetraacetic acid (EDTA), 0.5% (wt/vol) sodium deoxycholate, and 0.1% (wt/vol) SDS, 1 mM phenylmethylsulfonyl fluoride [PMSF]).
  5. Transfer the scraped cells into a 1-mL Dounce homogenizer placed on ice. Homogenize the cells by gently grinding them with the pestle.
  6. Incubate the homogenized cell lysates on ice for 10 min to ensure complete lysis. Save 20 µL of the total lysates to determine protein concentration by bicinchoninic acid (BCA) assay.
  7. Centrifuge the cell lysates at 13,500 × g at 4ºC for 10 min to pellet insoluble proteins.
  8. Carefully transfer the supernatant to a clean tube. Save 20 µL of supernatant to determine protein concentration by BCA assay.
  9. Concentrate the supernatant by performing methanol/chloroform precipitation.
  10. Resuspend the resulting precipitate in 0.1 mL of Laemmli sample buffer (25 mM Tris-HCl (pH 6.8), 1% (w/v) SDS, 12.5% (v/v) glycerol, 0.01% (w/v) bromophenol blue, 355 mM β-mercaptoethanol).
  11. Resuspend the pellet in Laemmli sample buffer and disperse it by sonication. 
  12. Perform three cycles of sonication at 0.5-s pulses at 10% power. Keep the probe tip toward the bottom of the tube to avoid frothing.
  13. Perform a bicinchoninic acid (BCA) protein assay on both the RIPA supernatant and the total cell lysates. 
  14. Load the protein samples onto a 12% or 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. Run the gel at a constant voltage of 200 V for 45 min.
  15. Transfer the proteins from the gel to a nitrocellulose membrane using a wet transfer system. Follow the manufacturer's instructions and transfer at 100 V for 70 min.
  16. Place the membrane in 5% (wt/vol) bovine serum albumin (BSA) dissolved in Tris-buffered saline with 0.1% (wt/vol) Tween-20 (TBST). Block for at least 1 h at room temperature with gentle shaking.
  17. Dilute the primary antibodies in TBST according to the manufacturer’s instructions. Incubate the membrane in the primary antibody solution overnight at 4ºC with gentle shaking.
  18. Rinse the membrane three times with TBST, each wash lasting 5 min.
  19. Dilute fluorophore- or horseradish peroxidase (HRP)-conjugated secondary antibodies in TBST at a 1:500 dilution (or as specified by the manufacturer).
  20. Incubate the membrane in the secondary antibody solution for 1 h at room temperature with gentle shaking.
  21. Rinse the membrane three times with TBST, each wash lasting 5 min.
  22. Develop the membrane using fluorescence or enhanced chemiluminescence (ECL) according to the manufacturer’s instructions.
  23. Visualize the protein bands using an imaging system.
    NOTE: To prevent protein degradation, perform all steps on ice or at 4ºC, except for antibody incubation and blocking steps. Ensure that all buffers, reagents, and equipment are pre-chilled before use to maintain low temperatures throughout the process. Avoid overloading the gel to prevent smearing, and optimize antibody concentrations and incubation times to suit the specific experiment.

6. Preparation of primary neurons for coculture

  1. Euthanize the pregnant rat using carbon dioxide as an inhalant anesthetic. Dissect out the uterus and place it in a sterile Petri dish.
  2. Remove the brains from the fetuses and place them in a 6-cm dish containing dissection solution. Ensure the tissue remains submerged in the solution at all times to prevent drying.
  3. Dissect the cortices and remove meninges as described (step 2.7).
  4. Transfer the dissected tissue to a laminar flow hood.
  5. Aspirate the dissection solution using a 1 mL pipette and add fresh dissection solution to the dish.  
  6. Mince the brain tissue into small pieces using a sterile scalpel blade.
  7. Transfer the minced tissue into 5 mL of neuronal dissociation medium (Table 1) pre-warmed to 37 ºC in a 15 mL tube.
  8. Incubate the tube in a 37 ºC water bath for 30 min, gently shaking the tube every 5 min to ensure proper enzyme-tissue interaction. 
  9. Remove the tube from the water bath and allow the tissue to settle at the bottom of the tube. Carefully aspirate the supernatant using a 1 mL pipette tip without disturbing the tissue.
  10. Add 5 mL of pre-warmed dissection solution to the tube and invert it 5 times to rinse the tissue. Let the tube stand for 3 min to allow the tissue to settle, then repeat the washing process three times, each time using fresh dissection solution.
  11. After the final wash, remove the supernatant and add 2.5 mL of dissection solution and 2.5 mL of plating medium.
  12. Triturate the tissue by pipetting the solution up and down 10–15 times using a 10-mL pipette.
  13. Allow the tube to stand for 3 min to let the tissue settle. Transfer the supernatant (containing cells) to a new 50 mL conical tube.
  14. Add 5 mL of plating medium to the original tube containing the settled tissue. 
  15. Triturate the tissue again 10–15 times using a 10-mL pipette until most chunks disappear.
  16. Pass the cell suspension through a sterile 40 µm cell strainer into a clean 50 mL conical tube containing 5 mL of plating medium.
  17. Centrifuge the filtered cell suspension at 200 × g for 5 min at room temperature.
  18. Remove the supernatant, then gently loosen the cell pellet by tapping the tube. Add 5 mL of pre-warmed plating medium and pipette up and down 10 times to resuspend the pellet.
  19. Count the cells as described previously (steps 2.21 and 2.22). Keep the cell suspension in a 37°C water bath during cell counting.
  20. Plate the neurons directly on top of the primary astrocytes in culture vessels as follows: for a 24-well plate, seed 1.45 × 105 cells per well; for a 6-well plate, seed 5 × 105 cells per well; for a 6-cm dish, seed 1 × 106 cells per well.
  21. Place the plates or dishes in a tissue culture incubator set to 37ºC with 5% CO₂. Avoid leaving the cells at room temperature for extended periods to maintain viability.
  22. Replace the plating medium with neuronal maintenance medium 4 h after plating.
  23. Replace half of the medium with fresh neuronal maintenance medium every 3–4 days.
    NOTE: The cocultured neurons will be ready for experiments at 7–10 days in vitro (DIV).

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Results

A morphological overview of primary astrocyte cultures at various time points after isolation is presented in Figure 3. Following the plating of the cortical cell suspension, some astrocytes adhere to the surface of the culture dish within the first 24 h (Figure 3A). During the initial 3 to 5 days, the cultures contain cell debris and dying neurons (Figure 3B), as the culture medium is optimized to support the survival and growth of...

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Discussion

Primary astrocyte cultures have significantly advanced our understanding of astrocytes' roles in health and disease. Building on foundational work by McCarthy and de Vellis21, these cultures are critical for studying AxD as they allow for detailed exploration of astrocyte-specific processes, particularly the effects of AxD mutations on GFAP assembly and aggregate formation.

The successful isolation and culture of primary astrocytes depend on several key steps that i...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank Dr. Tracy Hagemann (Waisman Center, University of Wisconsin-Madison) for providing the R237H knock-in rats. This work was supported by grants from the National Science and Technology Council (111-2320-B-007-008 and 112-2320-B-007-006 to N.-H. L. and M.-D.P.). The authors are grateful for the support from the confocal imaging core in National Tsing Hua University, Taiwan, which is supported by the National Science and Technology Council (NSTC-114-2740-M-007-001). We also thank the Instrument Center in the National Tsing Hua University for access to the electron microscope.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1 M HEPES , pH 7.4 Gibco15630-080
100× penicillin-streptomycin  Gibco15140-122
10× Hank's Balanced Salt Solution (HBSS)Gibco14180-046
2.5% TrypsinGibco15090046
30% Acrylamide/Bis solution 37.5:1Biorad1610158
anti-GFAP antibodyCell Signaling Technology12389
anti-human GFAP antibodyBiolegend837202Clone SMI21
anti-panGFAP antibodyAgilentGA524
anti-ubiquitin antibodyMilliporeST 1200Clone FK2
anti-vimentin antibodySigma-AldrichV6389Clone V9
anti-β III tubulin antibodyBiolegend801201
B27 supplementGibco17504044
Cacium chloride (CaCl2)Arcos34961
Cell culture-related reagents
ChemiDoc Imaging SystemBioRadNA
Coomassie blue dyeUSB32826
CysteineSigma-AldrichC1276
Dithiothreitol (DTT)URDTT
DNase I Sigma-AldrichD5025
Dounce homogenizerWheaton
Dulbecco's modified Eagle Medium (DMEM)Corning10-013-CM
Endotoxin-free plasmid DNA preparation kit Qiagen12943
Enhanced chemiluminescent (ECL) reagentPerkinElmerNEL 105001
Ethylenediaminetetraacetic acid (EDTA)USB15699
Fetal bovine serum  GibcoA5209401
GlucoseSigma-AldrichG8270
GlutaMaxGibco35050-061
Minimal essential medium (MEM)Corning10-010-CVR
NaClHoneywell31434S
Neurobasal mediumGibco21103049
Optima XE-90 UltracentrifugeBeckmanNA
PapainWorthingtonLS003126
Pnenylmethylsulfonyl fluoride (PMSF)ACROS ORGANICS215740050
PolyehtyleneimineSigma-AldrichP3143
Poly-L-lysineSigma-AldrichP6516
Potassium chloride (KCl)Honeywell12636
Rhodamine anti-actin antibodyBioRad12004164
Sodium dodecyl sulfate (SDS)USB75819
Sodium pyruvateGibco11360070
StarBright Blue 520 goat anti-mouse IgG Biorad12005867
StarBright Blue 700 goat anti-rabbit IgG BioRad12004161
SucroseSigma-AldrichS5391
SW-28 RotorBeckmanNA
TansIT-LT1 Transfection reagentMirusMR-MIR-2300
TEMEDUSB76320
Tris(hydroxymrthyl)aminomethane (Tris)USB75825
Triton X-100Sigma-AldrichT9284
β-mercaptoethanolSigma-AldrichM3148

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