1. Chapter 21 Quantitation Cell Counting Technique 1.Hemocytometer 2.Electronic Cell Counter

2. Hemocytometer 1.Counting chamber (optically flat chamber) 2.Hemocytometer Slide 3.Microscope 4.Cell suspension

3. Manual Cell Counting X100X400 Improved Neubauer

4. WBC Counting and RBC Counting Cultured Cell Counting = “WBC counting” 9 大格 25 中格 16 小格

5. http://biology.clc.uc.edu/fankhauser/Labs/Anatomy_&_Physiology/A&P202/Blood/Blood_Counts_practice.htm

7. 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)

8. Calculation 50 mL 1 mL 20 uL cell suspension Hemocytometer Assume: 28+30+35+56 1.Average = 37.25 2.Chamber volume =1x1x0.1 = 0.1 3.Cell number = (mm 3 )=1x10 -4 (cm 3 ) 200uL 15 mL 37.25 (cells)÷0.0001 (mL) x15 (mL) TOTAL cell number !!! Various Cell Density !!!

9. Attention 1.You have a single-cell suspension 2.It requires a minimum of 1x10 6 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

10. Electronic Cell Counter Beckman Scharfe System 1.Cell sizing 2.Discrimination between Viable and nonviable cells 3.Single cells and aggregates

11. 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

12. (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

13. 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 6. The calculated percentage of unstained cells will represent the percentage of viable cells. Cell Viability (within 1 min)

14. http://www.biomedcentral.com/1471-2202/6/40/figure/F1 Trypan blue exclusion assay

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