Chapter 21
Quantitation
Cell Counting Technique
1. Hemocytometer
2. Electronic Cell Counter
Hemocytometer
1. Counting chamber (optically flat chamber)
2. Hemocytometer Slide
3. Microscope
4. Cell suspension
Manual Cell Counting
Improved
Neubauer
X100
X400
WBC Counting and RBC Counting
9大格
25中格
16小格
Cultured Cell Counting = “WBC counting”
http://biology.clc.uc.edu/fankhauser/Labs/Anatomy_&_Physiology/A&P202/Blood/Blood_Counts_practice.htm
Calculation of cell numbers
1. Area = 1 mm x 1 mm
2. Height: 0.1 mm
3. Average of cell number
4. Calculation (cell numbers)
Calculation
50 mL
15 mL
1 mL
200uL
20 uL
cell suspension
Hemocytometer
Assume: 28+30+35+56
1. Average = 37.25
2. Chamber volume =1x1x0.1 = 0.1 (mm3) =1x10-4 (cm3)
3. Cell number = 37.25 (cells)÷0.0001 (mL) x15 (mL)
TOTAL cell number !!!
Various Cell Density !!!
Attention
1. You have a single-cell suspension
2. It requires a minimum of 1x106 cell/mL
3. Do not allow the cell time to settle or adhere in the tip of the pipette
before transferring them to the chamber
4. If cell aggregation can not be eliminated, lyse the cells in 0.1M
citric acid containing 0.1% crystal violet at 37C for 1 hour and then
count the nuclei
Electronic Cell Counter
1. Cell sizing
2. Discrimination between Viable
and nonviable cells
3. Single cells and aggregates
Beckman
Scharfe System
Electrical Resistance
1. As each cell passes through the
orifice, it changes the resistance to
the current flowing
2. The size of the pulse is proportional
to the volume of the cell
Automated Cell Viability Analysis
(a) Automation of the standard trypan blue assay
(b) % Viability
(c) Total cell concentration
(d) Total viable cell concentration
(e) Mean cell size
(f) Real time cellular images
Cell Viability
1. Prepare a cell suspension of the cells to be assayed
2. Prepare a 1:1 dilution of the suspension using a 0.4% trypan blue solution.
3. Load the counting chambers of a hemocytometer with the dilution.
4. Let sit for 1-2 minutes (do no leave longer as viable cells may die and begin to take up the
dye).
5. Count the number of stained cells and total number of cells (within 1 min)
6. The calculated percentage of unstained cells will represent the percentage of viable cells.
Trypan blue exclusion assay
http://www.biomedcentral.com/1471-2202/6/40/figure/F1
MTT ssay
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium