Date Issued: Jan 15, 2014
Global Pathogen Laboratory
Emerging pathogen Institute
University of Florida
SOP: Cell Passage
SOP: Cell Passage
One Health Center of Excellence
Emerging Pathogen Institute
University of Florida
I.
PURPOSE:
This protocol is to be used when there is a need to keep live cell lines under cultured
conditions for extended periods of time, results in the passaging/splitting of cell cultures.
II.
PRECAUTION:
Chemical Hazard:
Biological Hazard:


III.
Wear appropriate personal protective equipment at all times
All waste media and buffers should be treated as biohazard waste
PROCEDURE:
1. Dump media and rinse cells with 0.1 M PBS
2. Dump off PBS and rinse cells again with sterile 0.1 M PBS
3. Dump off PBS and use 5ml pipette to remove all residual PBS from each flask
4. Add trypsin to each flask:
MDCK Cells
T150
4 ml
T75
2 ml
T25
1 ml
2 ml
1 ml
0.5 ml
(use 0.05% Trypsin EDTA)
A549 Cells
(use 0.25% Trypsin EDTA)
5. Incubate in clean incubator for 10-15 min. Chech on cells after 10 min (MDCK) or 3
min (A549) and gently rock flask.
Date Issued: Jan 15, 2014
Global Pathogen Laboratory
Emerging pathogen Institute
University of Florida
SOP: Cell Passage
6. Add FBS-MEM media to neutralize trypsin:
MDCK Cells
T150
6 ml
T75
3 ml
T25
2 ml
18 ml
9 ml
4.5 ml
(use 5% FBS MEM)
A549 Cells
(use 10% FBS MEM)
7. Using a 5ml pipette, gently pipette up and down until no cell clumps are visible. A
cell scraper may be used to assure all cells are removed, followed by a PBS rinse.
8. Transfer cells to a 50 ml centrifuge tube
9. Obtain two 1.5 ml centrifuge tubes and add 1ml of PBS to one tube
10. Dilute cells 1:4 for passing cells (see microneut protocol for microneut cell prep)
a. To dilute 1:4, add 150 µl of PBS (from the first tube) to the second tube
11. Add 50 µl of cells and fill both sides of the slide to count
12. Count all four corners of the grid
13. To Count Cells:
a. (# cells / # grids counted) X dilution factor X 14 = cells/ml
14. To Seed Flasks:
a. Cell concentration desired / cell count/ml = ml of cells to add to each flask
b. Add appropriate media for cell type (T150 = 25ml, T75 = 12ml, T25 = 4ml)
APPENDIX I:
Materials:
APPENDIX II:
Reagents: