Counting Cells

Counting cells using trypan blue

Last updated: 1/12/12 By: Tim Starr

Overview

This protocol describes how to count cells using trypan blue. Trypan blue is normally excreted from live cells, so count only cells that appear clear in the microscope.

Protocol

• Start with 10 ml of media containing cells in a 15 ml conical.
• Shake tube to evenly disperse before removing aliquot
• In a 96-well plate, mix 10 ml of trypan blue with 10 ml of cells.
• Place 10 µl of trypan blue/cell mixture in the "V" of the hemocytometer slide, gently holding the coverslip on top

• You can count two samples at once, by placing another 10 µl of a different sample on the other side of the hemacytometer.
• Place slide on microscope with 10x magnification
• Count all the non-blue cells in the upper left hand section of the grid. Note: There are 9 sections in the hemacytometer grid (3 x 3), with each of the sections further sub-divided into smaller boxes to make it easier to count cells. The upper left section is divided into 16 smaller (4x4) boxes. Count all the cells in these 16 boxes. If there are too many cells to accurately count, use one of the middle sections that have more subdivisions to make counting easier.
• Rule of thumb for cells on the edge of the section, if the cell is more than half in the box count it, otherwise do not count it.

• If there are fewer than 100 cells in the upper left hand section, count another section (generally I go upper left, upper right, lower right, lower left). Continue counting sections until you have counted more than 100 cells. If you count two sections, divide the total by 2, if you count 3 sections, divide the total by 3, etc. This number is the # of cells in one section.
• Calculate the number of cells/ml using the following formula:

(# of cells in one section) x (trypan blue dilution factor) x 10,000 = # of cells/ml.

• The trypan blue dilution factor is 2 if you mixed 10 µl sample with 10 µl trypan blue.
• Note: If cells are too concentrated, can mix 10 µl sample with 90 µl trypan blue, count, but use a trypan blue dilution factor of 10.