hemocytocount v0.2

Haemocytometer counts

Enter live and dead counts for each 1 mm square you counted. Leave uncounted squares blank — the calculation uses only the squares with a value.

Live (unstained) Dead (stained) Count cells touching these lines

Tally clicker

Square

Sample

Results

Viability —

Tap a concentration to highlight it for copying, or type over it to use a value measured elsewhere (e.g. 1.2e6).

Total cells in suspension

Live cells —
Dead cells —
All cells —

cells/mL = (cells counted ÷ squares counted) × dilution factor × 104

Volume calculator — how much suspension for a given number of cells?
Live cells needed
× 10 cells
Volume of cell suspension to take —

Based on the live cell concentration above.

Plate seeding — media and cell volumes for wells, dishes or flasks

Option A — media first, then cells, into each well

1. Media per well —
2. Cell suspension per well —

Option B — dilute the cells first, then dispense

1. Cell suspension to take —
2. Media to add to it —
3. Mix, then dispense into each well —

Suggested volumes and seeding densities are from Thermo Fisher — Useful numbers for cell culture (densities are for HeLa cells; adjust for your cell type).

Instructions

Theory

A Neubauer haemocytometer is a counting chamber with an etched grid. Each large square is 1 mm × 1 mm and the chamber is 0.1 mm deep, so one large square holds 0.1 µL (10−4 mL). The mean count per large square multiplied by 104 therefore gives cells per mL of what was loaded; multiplying by the dilution factor gives the concentration of the original suspension.

Dye exclusion: live cells have intact membranes that exclude dyes such as trypan blue, so they look bright and clear. Dead cells take up the dye and look blue.

cells/mL = mean count per square × dilution factor × 104
viability (%) = live ÷ (live + dead) × 100

Which cells to count

Preparing the cell suspension

  1. Centrifuge the cells to form a pellet.
  2. Aspirate the media without disturbing the pellet.
  3. Resuspend the pellet in a known volume of media and mix to a single-cell suspension.
  4. Take a 10 µL aliquot of the cell suspension.
  5. Add it 1:1 to 10 µL of exclusion dye (e.g. 0.4% trypan blue) — a dilution factor of 2.
  6. Mix well by gentle pipetting.
  7. Transfer 10 µL into the haemocytometer chamber under the coverslip.
  8. View with a microscope and count promptly, as prolonged exposure to the dye is toxic to cells.

Using this app

Further reading: Trypan Blue Protocol · How to Count Cells with a Hemocytometer