TITLE: Cell counting with a haemocytometer (with mouse bone marrow example)
AUTHORS: Thomas Ashhurst, Darren A. Cox, Adrian L. Smith, Nicholas J. C. King

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div></div>

[STEPS]
?. [Counting cells]
Take 10 µL of this mixture and load onto the haemocytometer.
?. [Calculate concentration]
Calculate cells/sample using the following formula:
?. [Calculate concentration]
Determine required cell numbers and distribute into plateCalculate volume containing the required cell numbers: Vreq =  (Creq / Csample) x VsampleVol(required, mL) = (Cells required /Cells in sample, total) x Vol (sample total, mL)
Example: if we want 3x106WBCs from a bone marrow sample with 1.4x107WBCs in 1 mL of FACS bufferVreq= (Creq/ Csample) x VsampleVreq= (3x106cells / 1.4x107cells) x 1 mLVreq= 0.214 mL = 214 µL (i.e. 214 µL of sample contains 3x106WBCs)
?. [Prepare cell suspension]
Mix the cell suspension by inverting the tube numerous times, add 10 µL of the cell suspension to 40 µL of FACS buffer (1/5 dilution), then add 10 µL of this solution to 10 µL of 0.4% trypan blue (1/2 dilution factor), resulting in a final dilution factor of 1/50.
?. [Isolate cells]
Keep cell suspension on ice on in a refrigerator where possible.
InfoIt is established that some cellular markers, such as surface expression of M-CSFR (CD115), are downregulated when cells are removed from their biological niche. Storing cells at 4°C or on ice prevents this downregulation.
?. [Counting cells]
Place haemocytometer under the microscope and find the counting grid.
?. [Counting cells]
Count the total number of LIVE (clear) and DEAD (blue) leukocytes in the large centre square (“A”, blue in Figure 6A) which consists of 5x5 smaller squares (edges marked by triple lines in panel F), each of which contains 4x4 smaller squares (edges marked by single lines in panel F).
?. [Calculate concentration]
Using a pipette, transfer the necessary volume for a desired number of cells into relevant wells of a U- or V-bottom 96 well plate. For flow cytometry: typically stain 1 × 10^6 cells per sample.
?. [Counting cells]
Count the total number of erythrocytes in the same area, if desired.
?. [Counting cells]
If fewer than 100 total live leukocytes are counted in the centre square, then count the cells in the other large squares progressively, making sure each square is completely counted (B, C, D, E) until at least 100 live leukocytes have been counted in total.
If too few cells are present with this 1/50 dilution, then this process can be repeated at a higher concentration.