Assembling the Chamber
- Place the bottom plate on the work surface with registration pins and hardware pointing up. Place a 1mm thick 25 x 75mm glass microscope slide in the recess in the bottom plate. If you use a Neuro Probe slide with hydrophobic masking instead of a standard slide, position the corner of the slide with the Neuro Probe logo tightly into the corner of the chamber recess nearest the chamber’s engraved logo.
- Moisten the pre-punched filter paper with 0.15 M NaCl or PBS and blot briefly between paper towels. Align the holes in the filter paper with the pins on the bottom plate and lower the paper onto the microscope slide.
- Slide the silicone gasket onto the hardware with the registration holes over the registration pins. Align the top plate so that its NP logo matches the logo on the bottom plate and lower it onto the gasket. Apply and tighten the thumbnuts. The nuts should be finger tight only; do not use a wrench or pliers!
Adding and Settling the Cell Suspension
- Adjust the concentration of cells in the suspension so that 50µL of suspension contains the desired number of cells for one 32mm2 site. Concentrations of from 103 to 106 cells/µL may be used. Pipette 50µL of cell suspension into each of the eight wells.
- If the chamber is allowed to settle by gravity, it will take from 5 to 35 minutes for the liquid to wick away, leaving the settled cells in a monolayer on the dry slide. In order to recover virtually all the cells, spread in a random distribution suitable for differential counting, the filled chamber should be centrifuged. This is particularly important with cells of relatively low density, such as lymphocytes.
- Using the adapter and counterweight in the centrifuge accessory pack (Stock # DC8AP), place the filled chamber in a centrifuge equipped with a microplate carrier. Spin at 50 x g for 30 seconds.
- When the wells are dry disassemble the chamber and remove the microscope slide. If any moisture remains, allow the slide to air dry completely. Put the chamber in distilled or de-ionized water to soak.
- Fix the adherent cells with methanol or equivalent and stain with Diff-Quik (available from many major laboratory suppliers) or equivalent. If the cells do not readily adhere to glass, we recommend specially prepared Neuro Probe slides with enhanced binding properties. Cell sites on these slides are masked with a hydrophobic coating that restricts pipetted solutions to individual sites. Thus different solutions can be applied to different sites without contamination from one site to another.
- Place the slide under a microscope for morphological identification and differential counting. Used according to this protocol the DC8 settling chamber will permit virtually 100% recovery of cells, randomly distributed and sufficiently spread to exhibit excellent morphology.
- Packages of prepunched wicking filter papers (Stock # DC8WF1) and microscope slides with hydrophobic mask and enhanced binding properties (Stock # DC8SC) are available.
- Follow the instructions for cleaning and sterilizing your DC8 chamber. Reread and heed the warnings.
Leonard, Yoshimura, Skeel, and Goodwin. “A Multiwell Cell Settling and Adherence Chamber for Morphology and Differential Counting.” 1990, BioTechniques, 9 (6), 684-689.