This workflow is for labs that still count cells manually and need a clean path from hemocytometer data to a final plated suspension that can be dispensed consistently.
Steps
- Mix the harvested suspension and prepare the trypan blue dilution.
- Count enough squares to obtain a stable average and separate live from dead cells.
- Convert the counts into viable concentration and viability.
- Decide the target density for the plate format and assay timing.
- Build one mixed suspension with enough overage for the full plate.
- Re-mix immediately before dispensing each set of wells.
- Record passage number, morphology notes, and the final seeding plan.
Control points
- Weak count quality creates weak seeding accuracy.
- Plate area and treatment timing matter as much as the raw cell number.
- If the suspension settles quickly, the dispensing order can bias the plate.
Use these tools
- Hemocytometer
- Cell Seeding
- Cell Doubling Time
Tips
- Repeat the count if square-to-square spread looks implausibly high.
- Avoid recalculating per well at the bench; prepare one shared suspension instead.
- Pair the seeding note with expected confluence timing so future runs can be tuned.