Purpose: Subculture, passage and count cells using Trypan Blue.

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Materials: 

(2) 1.5 ml Epindorf tubes

Micropipettes: P20, P200, P1000

2 ml Pipette

10 ml Pipette

Pipette Aid

T25 Flask

5 mls PBS

Media DMEM F12

Trypan Blue: Fluka Lot 1151527 24704PO5 Exp 05/2007

1 ml Trypsin

Hemacytometer

10% Bleach Solution



Methods: 



• Removed T25 cell flask from Incubator
• Observed cells using inverted microscope (approximately 100% confluent and somewhat vacuolated) 
• Removed and disposed of spent media in 10% bleach waste solution
• Added 5 mls of PBS and carefully rotated the flask (flatly) (N,S,E,W)
• Removed and disposed of PBS in waste solution
• Added 1 ml of Trypsin 
• Replaced flask in 37 degree C incubator  for approximately 3 minutes
• Observed cells again using inverted microscope (detached)
• Added 9 mls of media
• Triturated and observed cells again (Not in single cell suspension so continued to rinse and triturate for approximately one more minute)
• Observed cells in inverted microscope again (cells in single cell suspension)
• Used P1000 to remove 0.5 ml aliquot and placed in 1.5 ml epi tube
• Triturated again
• Removed 0.1 ml aliquot and transferred to new epi tube. 
• Added 0.1 ml Trypan Blue to 0.1 ml in 2nd epi tube and carefully triturated
• Loaded hemacytometer with 10 microliters per side using P20
• Observed in microscope

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Results: 



Count

17,

18, 

18, 

(4th quad was almost empty - not used)



Used average of 3. 



17.67 cell average x 2 (dilution factor) x 10,000



3.53 x 10^5/1ml = 2 x 10^5/x   x=0.567 mls



Plated 2x10^5 cells in new flask. 1:20 split in old flask



Each flask had 0.6mls of cells and 9.4 mls of media.