## Bart's Cookbook-- HOW TO USE THE COULTER COUNTER TO COUNT CELLS

1) Turn on the counter by pulling out the on/off button. You need to do this at least 10 min before use to obtain sufficient vacuum.

Usually put 0.2 ml of your suspension of cells into 10 ml Tris or TD. In reality you can use any dilution, as long as you know how to multiply.

2) Be sure to mix the sample thoroughly before counting, since the cells will settle to the bottom. To count the sample, turn the glass valve directly above the cells to vertical (open). Wait until you see a green baseline in the "sizing" monitor--the vertical "blips" represent either cells or crud. The larger the blip, the larger the cell. Turn valve A back to horizontal (closed) to begin the count.

The length of the count should be approx. 13 sec, from when the first number appears in the register to when the green display goes out. If the counting time is greater than 13 sec, the aperture is clogged with debris and should be cleaned gently with the brush provided (as well as "flushed," see 5). Any count which is obtained when the aperture is obstructed or downright clogged has an unpredicatable coincidence and should be done again.

The count displayed is the the number of cells in 0.5 ml. If you diluted 0.2 ml of cells to 10 ml, multiply the number displayed by 100 to determine the number of cells per ml in the original culture. Be sure to use the coincidence correction table if your count is greater than 10,000. At high concentrations the probability of two or more cells passing through the aperture simultaneously, and being counted as one cell, is high.

Find the first three digits of your count on the table and use the corrected figure, which is next to it, for your calculations. Obviously, if the cell count displayed is too low or too high (less than 3000 or greater than 99,900 cells per 0.5 ml), you should set up an appropriate dilution and re-count the sample.

3) When you're finished counting, flush the system. Put the electrode into the vial containing Isoterge and check to see that the flask labeled "Rinse Flask" contains at least 100 ml of solution. If it's low, fill it with Tris or TD and some Isoterge. Turn valve A to vertical, then turn valve B, which is off to the left, to vertical. Flushing will occur. After 10 sec, turn valve B back to horizontal and then turn valve A to horizontal. Store the electrode in the vial of Isoterge, and, if it's the end of the day, turn the machine off.