As part of assay development and optimization, it is essential to evaluate key experimental parameters such as cell density and extracellular matrix (ECM) coating conditions. Based on our internal work and collaborations with customers using a wide range of neuronal cell types, we have consistently observed that a combination of poly-D-lysine (PDL) and laminin yields the most reliable neuronal attachment and growth on CytoTronics’ microplates!
In this protocol, we share our recommended coating method for PDL and laminin. This protocol is not intended to replace any coatings you may already be using successfully; rather, we encourage all customers to include PDL/laminin as one of the coating conditions tested during Pixel assay optimization.
As best practice, we also recommend plating cells in parallel on a sister glass-bottom dish under the same conditions to monitor cell health and morphology independent of measurements.
Materials
Poly-D-lysine (Thermo Fisher, Cat No. A389-401)
Laminin (Thermo Fisher, Cat No. 23017015)
Phosphate-Buffered Saline (PBS)
Sister glass-bottom dish (Fisher Scientific, Cat No. NC0536760)
Multichannel pipette options:
Manual: Corning Lambda EliteTouch Multichannel Pipettor
Electronic: VIAFLO Lightweight Electronic Multichannel Pipettors
Electronic whole plate: VIAFLO96
Multichannel vacuum aspirator
Note: Once cells have been plated, we recommend using an electronic pipette with adjustable flow speed and operating it at the slowest setting to minimize disturbance to the attached cells. To further reduce disruption, perform half-media exchanges rather than full media changes.
Protocol
All steps in this protocol should be performed using sterile technique within a biosafety cabinet to maintain sterility and prevent contamination.
Prior to beginning the coating protocol described here, rehydrate the microplate accorded to the Electrode Rehydration instructions, and perform a Calibration Scan to assess the overall quality of the plate and electrodes. For detailed guidance on these steps, please refer to the User Guide.
Note: Once the wells have been rehydrated, take care to not let the wells dry out – all subsequent solution changes should be performed promptly to maintain electrode integrity.
Coat with Poly-D-Lysine (PDL)
Add 50 μL of PDL (0.1 mg/mL) to each well of the microplate.
Note: For the sister glass-bottom dish (in which the wells are approximately twice the surface area of CytoTronics' microplate), add 100 μL per well, or ensure that the 50 μL fully covers the entire well bottom.
Incubate Overnight
Seal the plate with Parafilm to prevent evaporation.
Incubate the plate at 4°C overnight to allow PDL to bind to the surface.
Wash Thoroughly
The following day, aspirate the PDL solution using a multichannel vacuum aspirator and wash each well three times with 150 μL of PBS.
Note: If a multichannel vacuum aspirator is not available, use a multichannel pipette to carefully aspirate all solution from each well. Proceed immediately to the next solution step to ensure the electrodes do not dry out.
Note: Residual PDL can be cytotoxic to neurons. Ensure all PDL is completely removed by washing thoroughly.
Prepare Laminin Solution
Dilute laminin in PBS to a final concentration of 20 μg/mL (approximately 1:60 dilution, depending on stock concentration).
Example: If the stock is 1.2 mg/mL, dilute 100 μL of stock laminin into 6 mL of PBS. This volume will coat one full microplate and part of a glass-bottom dish at 50 μL per well.
Coat with Laminin
Add 50 μL of the diluted laminin solution to each well after aspirating the final PBS wash.
Incubate at 37°C for 1 hour.
Prepare for Plating
While the laminin incubates, thaw and prepare your cells for plating.
After incubation, aspirate the laminin with a multichannel vacuum aspirator and immediately plate cells into each well.
Note: Do not wash after laminin coating and avoid letting wells dry out between laminin removal and cell plating.
Tip: To promote even cell distribution across each well, allow the microplate to sit undisturbed in the biosafety cabinet for 10-20 minutes after plating the cells.