Cell Counting
1Thompson Children's Hospital, Chattanooga, Tennessee
2Purdue University, West Lafayette, Indiana
Abstract
This unit presents protocols for counting cells using either a hemacytometer or electronically using a Coulter counter. Cell counting with a hemacytometer permits effective discrimination of live from dead cells using trypan blue exclusion. In addition, the procedure is less subject to errors arising from cell clumping or size heterogeneity. Counting cells is more quickly and easily performed using an electronic counter, but live-dead discrimination is unreliable. Cell populations containing large numbers of dead cells and/or cell clumps are difficult to count accurately. In addition, electronic counting requires resetting of the instrument for cell populations of different sizes; heterogeneous populations can give rise to inaccurate counts, and resting and activated cells may require counting at separate settings. In general, electronic cell counting is best performed on fresh peripheral blood cells.
Materials
Basic Protocol 1: Counting Cells Using a Hemacytometer
- 70% (v/v) ethanol
- Cell suspension
- 0.4% (w/v) trypan blue or 0.4% (w/v) nigrosin, prepared in HBSS (appendix 2A)
- Hemacytometer with coverslip (e.g., Improved Neubauer, VWR)
- Hand-held counter
Basic Protocol 2: Counting Cells Using a Coulter Counter
- Cell suspension
- 20 ml counting vials
- Zap-O-globin (Coulter), lysing and hemoglobin reagent (VWR), or equivalent (for counting white blood cells)
Figures
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Figure A.3A.1Hemacytometer slide (Improved Neubauer) and coverslip. Coverslip is applied to slide and cell suspension is added to counting chamber using a Pasteur pipet. Each counting chamber has a 3 × 3mm grid (enlarged). The four corner squares (1, 2, 4, and 5) and the central square (3) are counted on each side of the hemacytometer (numbers added).
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