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

X100 X400

ImprovedNeubauer

WBC Counting and RBC Counting

Cultured Cell Counting = “WBC counting”

9大格

25中格

16小格

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

1 mL

20 uLcell suspension Hemocytometer

Assume: 28+30+35+56

1. Average = 37.252. Chamber volume =1x1x0.1 = 0.1

3. Cell number =

(mm3) =1x10-4 (cm3)

200uL

15 mL

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 pipettebefore 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 thencount the nuclei

Electronic Cell Counter

Beckman

Scharfe System

1. Cell sizing

2. Discrimination between Viableand nonviable cells

3. Single cells and aggregates

Electrical Resistance

1. As each cell passes through theorifice, it changes the resistance tothe current flowing

2. The size of the pulse is proportionalto the volume of the cell

(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

Automated Cell Viability Analysis

1. Prepare a cell suspension of the cells to be assayed2. 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 6. The calculated percentage of unstained cells will represent the percentage of viable cells.

Cell Viability

(within 1 min)

http://www.biomedcentral.com/1471-2202/6/40/figure/F1

Trypan blue exclusion assay

MTT ssay

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium