Neural Precursor Cell-Based Screening & Bioassay Kit

Discontinued Product

SC014 has been discontinued.
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Neural Precursor Cell-Based Screening & Bioassay Kit Summary

Specifications

Storage
Store the unopened product at 2 - 8 °C. Do not use past expiration date.

Product Datasheets

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
 

Reagents Provided

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.

Other Supplies Required

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
 

Procedure Overview

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

    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
  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.
  3.  

    After 24 hours, add 20 µL of fresh media per well
  1. After 24 hours, add 20 µL of fresh media per well.
  2.  

    48 hours after initial cell plating, add 20 µL of fresh media and 24 µL of Resazurin 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.
  3.  
  1. Assess cell proliferation by reading fluorescence using 544 nm excitation and 590 nm emission wavelengths.

NPC Differentiation Assay

    Coat wells of a 96-well plate with Poly-L-Ornithine and Fibronectin
  1. Coat wells of a 96-well plate with Poly-L-Ornithine and Fibronectin.
  2.  

    Plate 2.5 - 5.0 x 10<sup>4</sup> NPCs/well in 200 µL of Maintenance Supplemented Media
  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.
  3.  

    After 24 hours, add 100 µL of fresh media per well.
  1. After 24 hours, add 100 µL of fresh media per well.
  2.  

Day 0 of Differentiation

    48 hours after initial cell plating, replace the media with Differentiation Supplemented Media +/- vehicle control or bioactive agent of interest
  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.
  3.  

Day 2,4, & 6 of Differentiation

    Replace the media with fresh Differentiation Supplemented Media +/- vehicle control or bioactive agent of interest
  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.
  3.  

Day 7 of Differentiation

    Fix cells and perform a cell-based ELISA using an HRP-conjugated Neuron-specific beta-III Tubulin antibody
  1. Fix cells and perform a cell-based ELISA using an HRP-conjugated Neuron-specific beta-III Tubulin antibody.
  2.  

FAQs

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