Trypan Blue Staining: Viability Cell Count

Materials:

• Suspension culture of cells
• Sterile transfer pipettes Stock 0.2% (w/v)
• Trypan blue
• Hemacytometer and microscope

Protocol:

1. Gently swirl a suspension culture to distribute the cells evenly. Aseptically remove a small sample (0.1 mL) of cells from the cultures. Place the sample in a separate test tube (it need not be sterile).
   
2. Dilute 4 parts of stock Trypan Blue with 1 part of 5X saline and add 0.1 mL of the diluted dye to your sample. Mix gently.
   
3. Set up a hemocytometer and cover slip. Immediately place a drop of the stain/culture combination on the hemocytometer (remember to use both sides of the hemocytometer) and wait one minute.
   
4. Observe the cells with low power microscopy. Count the total number of cells, and the number of stained cells.
   
5. Compute the concentration of viable cells per mL of culture.

Notes: Trypan Blue is a stain that is actively extruded from viable cells, but which readily enters and stains dead cells. Therefore, the cells which are blue are dead. The difference between the total number of cells and the number of dead cells would be the number of viable cells in a given aliquot of your culture.

Marcatura con Sytox green

The SYTOX green dye is impermeant to live cells and apoptotic cells, but stains necrotic cells with intense green fluorescence by binding to cellular nucleic acids.

SYTOX Green nucleic acid stain (S7020) is particularly useful for cell-cycle analysis on RNase-treated fixed cells

Protocollo di marcatura:

• lavare le cellule con PBS
  
• fissare le cellule in PAF 4% per 20' (questa fase del protocollo deve essere eseguita sotto cappa chimica)
• lavare le cellule con PBS
  
• incubare le cellule con PBS-Triton 0.3% per 15'
  
• incubare con Sytox Green diluito 1:30.000 in PBS per 10'-15'
  
• lavare tre volte con PBS
• montare
  

Marcatura con Phalloidin

• lavare le cellule con PBS
• fissare le cellule in PAF 4% per 20' (questa fase del protocollo deve essere eseguita sotto cappa chimica)
• lavare le cellule con PBS
  
• incubare le cellule con PBS-Triton 0.3% per 15'
  
• incubare le cellule con Rhodamine-phalloidin (R415) diluita 1:20 in PBS per 20'
  
• lavare tre volte con PBS

*** Phalloidin binding requires the F-actin to have a protein structure near native. Methanol or acetone used to fix and / or permeabilize essentially abolishes phalloidin binding. ****