Neural Precursor Cell-Based Screening & Bioassay Kit

Assay Procedure

Refer to the product datasheet for complete product details.

Briefly, the effects of compounds on neural progenitor cell (NPC) proliferation and differentiation can be evaluated using the following in vitro procedure:

• Culture NPCs with bioactive agents of interest
• Assess NPC proliferation using a Redox-sensitive dye
• Evaluate NPC differentiation using a cell-based ELISA

View Graphic Procedure

 

Reagents supplied in the Neural Progenitor Cell-Based Screening & Bioassay Kit (Catalog # SC014):

• Maintenance Supplement (500X)
• Differentiation Supplement (100X)
• Fibronectin (100X)
• Resazurin
• Wash Buffer (10X)

• Blocking Buffer
• HRP-conjugated Mouse Anti-Neuron-specific  beta-III Tubulin
• Concentrated HRP substrate and diluent
• Plate sealers

Note: The reagents provided in the kit are sufficient for two 96-well plate proliferation assays and two 96-well plate differentiation assays.

Reagents

• NPC medium (e.g., N-2 Plus Media Supplement (Catalog # AR003) or N-2 MAX Media Supplement (Catalog # AR009) in DMEM or equivalent)
• Poly-L-Ornithine
• Phosphate Buffered Saline (PBS)

• Penicillin-Streptomycin-Glutamate (100X)
• 4% Paraformaldehyde in PBS
• Sterile deionized water
• 30% H₂O₂
• 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI)

Materials

• NPCs (Rat Cortical Stem Cells; Catalog # NSC001 or equivalent)
• 15 mL centrifuge tubes
• Pipettes and pipette tips
• Serological pipettes

• Reagent reservoirs
• 96-well cell culture microplates
• Black 96-well clear-bottom cell culture microplates
• 96-well round bottom plates

Equipment

• 37 °C and 5% CO₂ incubator
• Centrifuge
• Hemocytometer
• Inverted microscope

• 37 °C water bath
• Horizontal orbital microplate shaker
• Multi-channel pipette
• Fluorescence microplate reader

This protocol has been tested using rat cortical stem cells. If using a different cell line, the protocol below may need to be optimized.

NPC Proliferation Assay

1. Plate 1.3 – 2.5 x 10⁴ NPCs/well in 100 µL of Maintenance Supplemented Media +/- 100 µL vehicle control or bioactive agent of interest.
2. Incubate the cells at 37 °C and 5% CO₂.
 

---


1. After 24 hours, add 20 µL of fresh media per well.
 

---


1. 48 hours after initial cell plating, add 20 µL of fresh media and 24 µL of Resazurin per well.
2. Incubate the cells at 37 °C and 5% CO₂.
 

1. Assess cell proliferation by reading fluorescence using 544 nm excitation and 590 nm emission wavelengths.

NPC Differentiation Assay

1. Coat wells of a 96-well plate with Poly-L-Ornithine and Fibronectin.
 

---


1. Plate 2.5 - 5.0 x 10⁴ NPCs/well in 200 µL of Maintenance Supplemented Media.
2. Incubate the cells at 37 °C and 5% CO₂.
 

---


1. After 24 hours, add 100 µL of fresh media per well.
 

Day 0 of Differentiation

1. 48 hours after initial cell plating, replace the media with Differentiation Supplemented Media +/- vehicle control or bioactive agent of interest.
2. Incubate the cells at 37 °C and 5% CO₂.
 

Day 2,4, & 6 of Differentiation

1. Replace the media with fresh Differentiation Supplemented Media +/- vehicle control or bioactive agent of interest.
2. Incubate the cells at 37 °C and 5% CO2.
 

Day 7 of Differentiation

1. Fix cells and perform a cell-based ELISA using an HRP-conjugated Neuron-specific beta-III Tubulin antibody.