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URL: https://dx.doi.org/DOI:10.17504/protocols.io.btqgnmtw

Authors: Thomas Ashhurst, Darren A. Cox, Adrian L. Smith, Nicholas J. C. King

Group: Immune Dynamics

Summary: Cell counting is a critical step to determine the number of live leukocytes (white blood cells, WBC) and erythrocytes (red blood cells, RBC) per femur, and is accurately performed using a microscope and haemocytometer. This step allows enables the distribution of a specific number of cells per well in the staining plate. Some researchers prefer to perform an erythrocyte (or red blood cell, RBC) lysis prior to counting, as some researchers are concerned about misidentifying erythrocytes as leukocytes during counting. This RBC lysis removes all erythrocytes prior to counting, resulting in easier counting of leukocytes. However, we have found that differentiating leukocytes from erythrocytes is straight forward at a higher magnification based on size (erythrocytes are smallest), colour (erythrocytes are red in colour), and birefringence (leukocytes exhibit birefringence, erythrocytes do not). Additionally, RBC lysis prior to cell staining will have a small impact on CD115 staining levels (see protocol notes). To circumvent the need for RBC lysis prior to staining, we count the number of leukocytes (and erythrocytes) in the centre square only, at a high magnification. Alternatively, an automated cell counter can be used to speed up counting. However, care must be taken to ensure that any automated cell counter is accurately performing counts, as most automated cell counters are calibrated to human blood cells.

Affiliations: The University of Sydney, The University of Sydney, The Centenary Institute, The University of Sydney

External URL: https://link.springer.com/protocol/10.1007/978-1-4939-9454-0_12

Version: 1

Publication Date: 2021

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