Cell Preparation:

1. Prior to cell seeding for assay, culture neurons/astrocytes in growth media until ~70-80% confluent (unless cell seeding directly from thaw or isolation).
2. Detach cells from culture flasks/plates via method appropriate for cell type of interest. If necessary, coat assay plate wells with poly-D-lysine or extracellular matrix protein to enhance cell adhesion. Co-cultures of astrocytes and neurons may be seeded simultaneously or sequentially. For sequential seedings, plating of astrocytes first as a "basal" layer is recommended, followed by neuronal plating and differentiation, if necessary. Adjust cell density as appropriate for cell type, subsequent culture time, parameters of interest, etc. Depending on culture age, cell source and seeding density, primary cultures may vary greatly in rate of proliferation, GFAP expression or neurite outgrowth – it is important to characterize and optimize your cell system to provide the most biological relevance for your experimental model, as well as to provide for effective imaging, segmentation and analysis using HCS (see Figure 1). After adding cells to plate (cells may be seeded in 90 μL media, to facilitate toxin treatment as described in Step 3 below), allow plate to sit on a level surface at room temperature for 15-30 min, enabling even cell distribution. Following this period, incubate cells in growth media (37°C/5% CO₂) for at least 24 hours, then switch to differentiation culture conditions (e.g., low serum/NGF for PC12 cells), as appropriate. Continue culture until cells reach desired level of confluence or differentiation, changing media at intervals appropriate for cell type.
3. Cell treatments (control compounds, test compounds, etc.) can be introduced at any point during this culture period, as appropriate for time-course of treatment of interest. Acrylamide, hydrogen peroxide and K-252a are provided as neurotoxic control compounds. Sufficient reagents are provided for duplicate 12-point dose response curves (including one dH₂O or DMSO-control set within the dose response) for all five 96-well plates. The compounds are provided at 250X concentration (for K-252a, assuming a maximum treatment of 1 µM) or 10X (for acrylamide and hydrogen peroxide, assuming maximum treatments of 100 mM and 10 mM, respectively). Recommended treatment preparation involves half-log (1:√10) serial dilution of the 250X compound in DMSO, followed by dilution in Compound Dilution Buffer to 10X (10X control compounds originating in dH₂O may be serially diluted directly in sterile dH₂O or Compound Dilution Buffer). 10 µL of each treatment may then be added to the 90 µL of culture media already present in each well, for a final 1X concentration (0.4% DMSO or 10% dH₂O). Sample data is provided for 24 or 96 hours of compound treatment at 37°C prior to fixation.

Cell Fixation and Immunofluorescent Staining:

Note: Staining time is ~2.5 hours post-fixation. Do not allow wells to dry out between staining steps. Aspiration and dispensation of reagents should be conducted at low flow rates to diminish any cell loss due to fluid shear. All recommended ‘per well’ volumes refer to a single well of a 96-well microplate. All recommended ‘per 96-well plate’ volumes include 25% excess for liquid handling volume loss. All staining steps are performed at room temperature (RT). All buffers and antibody solutions are stable for at least 24 hours at RT.

4. At end of culture period, pre-warm HCS Fixation Solution (2X) to room temperature (RT) or 37ºC if desired (12 mL/96-well plate). In a chemical fume hood, add 100 µL/well directly to culture media and allow to fix for 30 min at RT. Remove fixative/toxin-containing media and dispose of in compliance with regulations for hazardous waste (see MSDS). If proceeding immediately to staining, rinse each well twice with 200 µL of HCS Immunofluorescence Buffer. Alternatively, if plates are to be stained at a later time, rinse twice with 200 µL of Wash Buffer, then leave second rinse volume in wells and store plates tightly sealed at 4ºC until staining.
5. If fixed samples have been stored at 4ºC prior to staining, rinse twice with 200 µL HCS Immunofluorescence Buffer before proceeding with staining protocol.
6. Prepare working solution of Rabbit Anti-βIII Tubulin/Mouse Anti-GFAP HCS Primary Antibodies (6 mL/96-well plate) as follows: Add 60 µL of each thawed primary antibody to 5.88 mL of HCS Immunofluorescence Buffer. Mix well. Remove previous Immunofluorescence Buffer rinse. Add 50 µL of Primary Antibody solution to each well and incubate for 1 hour at RT.
7. Remove Primary Antibody solution. Rinse three times with 200 µL HCS Immunofluorescence Buffer.
8. Prepare working solution of HCS Secondary Antibody/Hoechst HCS Nuclear Stain (6 mL/96-well plate) as follows: Add 30 µL of each thawed secondary antibody and 30 µL of thawed Hoechst HCS Nuclear Stain to 5.91 mL of HCS Immunofluorescence Buffer. Mix well, protecting solution from light. Remove previous HCS Immunofluorescence Buffer rinse. Add 50 µL of HCS Secondary Antibody/Hoechst HCS Nuclear Stain solution and incubate for 1 hour at RT, protected from light.
9. Remove HCS Secondary Antibody/Hoechst HCS Nuclear Stain solution. Rinse twice with 200 µL HCS Immunofluorescence Buffer.
10. Remove previous HCS Immunofluorescence Buffer rinse. Rinse twice with 200 µL of HCS Wash Buffer, leaving second rinse volume in wells.
11. Seal plate and image immediately, or store plate at 4ºC protected from light until ready for imaging.

Image Acquisition and Analysis:

12. Imaging and analysis of stained plates may be performed upon a variety of available HCS platforms, including the IN Cell Analyzer (GE Healthcare), ArrayScan (ThermoFisher Scientific) or Opera (Perkin Elmer). Some guidelines for imaging and analysis are provided in Table 1:

Table 1. Image Acquisition and Analysis Guidelines – HCS222 βIII-Tubulin/GFAP (Co-Culture) Assay

Representative Results:

Figure 1. βIII-tubulin and GFAP immunofluorescence of mixed rat neural cell cultures.

Co-cultures of primary rat hippocampal astrocytes with either primary rat hippocampal neurons or rat PC12 cells were generated by pre-plating astrocytes for several days in culture, followed by neuronal seeding. Primary neurons were cultured for an additional 11 days, while PC12s were cultured for an additional 2 days under differentiation conditions (low serum/NGF). Cell handling, fixation and immunostaining were performed according to HCS222 assay protocols. Cells were imaged on the GE IN Cell Analyzer 1000 (3.3) at 20X (primary neurons) or 10X (PC12) objective magnification and analyzed using the GE IN Cell Analyzer 1000 Workstation (3.4) Neurite Outgrowth and Multi Target Analysis algorithms. Top panel: Fused images of Hoechst HCS Nuclear Stain (blue), βIII-tubulin (green) and GFAP (red) fluorescence. Separate analysis of the βIII-tubulin fluorescence channel allows for neurite outgrowth segmentation (middle panel: cell bodies outlined in blue (primary neurons) or yellow (PC12), neurites in green (primary neurons) or light blue (PC12)). Analysis of the GFAP channel allows for astrocyte segmentation (bottom panel: nuclei outlined in blue, cytoplasm in green). GFAP segmentation for the primary rat hippocampal neuron/astrocyte co-culture demonstrates the ability to distinguish between nuclei/cell bodies that are GFAP (+) (green outlines) and those that are GFAP (-) (red), enabling separate cell counts for neurons and astrocytes in a mixed culture setting.


Figure 2. Dose responses of primary rat hippocampal neuron/astrocyte co-cultures to toxic stresses.

Primary rat hippocampal astrocytes (P6) were plated on 96-well plates in growth media and cultured for 6 days. Primary rat hippocampal neurons (direct from thaw) were then seeded on top of pre-plated astrocytes and cultured in growth media for 11 days. Cells were treated with serial dilutions of acrylamide or hydrogen peroxide (max. concentrations = 100mM and 10mM, respectively) for the last 24 hours of culture. Cell handling, fixation and immunostaining were performed according to HCS222 assay protocols. Cells were imaged on the GE IN Cell Analyzer 1000 (3.3) at 20X (10 fields/well) and analyzed (nuclear/cytoplasmic/neurite segmentation) using the GE IN Cell Analyzer 1000 Workstation (3.4) Neurite Outgrowth and Multi Target Analysis algorithms. Data presented are mean ± SEM, n = 3. Multiple parameters (neurite outgrowth, GFAP intensity, astrocyte area and neuronal or astrocytic cell counts) were investigated for dose-dependent trends.