labmatetools / 5.1 CELL SEEDING ALL TOOLS

Cell Count & Seeding

From hemocytometer squares to live cells/mL, then exactly how much cell suspension and medium to seed your plate, dish or flask.

1 · COUNTSECTION 05 — CELLS & PROTEIN · 5.1

I HAVE
A · COUNT THE CORNER SQUARESEACH 1 × 1 MM = 0.1 µL

Square 1 is enough. Squares 2–4 can be skipped — the mean uses the ones you fill.

SQ 1START HERE
SQ 2OPTIONAL
SQ 3OPTIONAL
SQ 4OPTIONAL
0 of 4 squaresGOOD RANGE 20–100
B · HOW YOU DILUTED THE CELLS
OFF
2 · TRYPAN BLUE MIX ×2
µL cells + µL trypan blue
TOTAL DILUTION×2
mL of cell suspension

2 · SEEDCOUNT → SEED → PLATE

▦PLATE OR FLASK

nCELLS TO SEED

Scientific notation works: 5e4 = 50,000

#WELLS / VESSELS
wells
VMEDIUM PER WELL
+EXTRA
%
MIX

SEEDING MIX
  1. CELL SUSPENSION—
  2. MEDIUM—
  3. TOTAL—
  4. PER WELL—
  5. CELLS PER WELL—
ABOUT THIS TOOL

How to use

  1. Mix your cells with trypan blue and load the hemocytometer.
  2. Count the live (clear) cells in a large corner square and type the number into that square on the drawing. Square 1 is enough; fill squares 2–4 too for a better average, or skip them.
  3. Add the dead (blue) cells in the same squares if you want viability.
  4. Enter how you mixed the cells with trypan blue — 10 µL cells + 10 µL dye is ×2. Diluted a dense culture before that? Turn on Pre-dilution and enter those volumes too; the two dilutions are multiplied.
  5. Already have a count from an automated counter? Choose A CELL COUNT and type it in.
  6. Pick your plate or flask, how many cells per well (or per cm²), how many wells, and the medium volume per well.
  7. Mix the cell suspension and medium as shown, then dispense the per-well volume. Mix the tube gently between wells so cells don't settle.

The formula

cells/mL = mean per square × dilution × 10⁴
dilutionpre-dilution × trypan blue mix, e.g. (10 + 10) ÷ 10 = 2
10⁴one large square holds 0.1 µL (1 mm × 1 mm × 0.1 mm)
viabilitylive ÷ (live + dead)
suspensioncells needed ÷ live cells/mL

Example. 52, 48, 50, 55 live in 4 squares, 1:1 with trypan blue: 51.25 × 2 × 10⁴ = 1.025 × 10⁶ cells/mL. Diluted 1:10 in PBS first: × 20 instead, 1.025 × 10⁷ cells/mL.

Good to know

  1. Free to use. No subscription. Nothing you type leaves your browser.
  2. Aim for roughly 20–100 cells per square; outside that, dilute or concentrate and recount.
  3. Plate areas and volumes are typical values — check your vessel's data sheet and your protocol.
  4. Seeding numbers use live cells only.
QUESTIONS · CELL COUNT & SEEDING CALCULATOR

Frequently asked

How do I calculate cells/mL from a hemocytometer?

cells/mL = mean count per large square × dilution × 10⁴. For 52, 48, 50 and 55 live cells mixed 1:1 with trypan blue: 51.25 × 2 × 10⁴ = 1.025 × 10⁶ cells/mL.

How is viability calculated?

Viability = live ÷ (live + dead). Count the blue (dead) cells in the same squares as the live ones.

I already have a count from an automated cell counter. Can I use it?

Yes. Choose A CELL COUNT and type the concentration in.

How many cells should I count per square?

Roughly 20–100 cells per square. Outside that range, dilute or concentrate the cells and count again.