Author: Unkown. Date: 1/5/95

Standard Operating Procedure:

FACS analysis of murine cells

Hazards:

SODIUM AZIDE: highly toxic. May cause heritable genetic damage. Very toxic by inhalation, in contact with skin, and if swallowed. heating may cause explosion. Avoid contact with metals. Do not brethe dust. Do not get solutions in eyes, on skin, or on clothing.

FITC, Phycoeurithrin,TRI-color, and BIOTIN: small quantities of conjugated chemicals are present in the antibody preparations. Avoid contact with eyes, skin and clothing.

Protection:

Lab coat

gloves

Wear a dust mask when working with dry sodium azide

Waste:

drain

Spill Clean-Up:

 

Procedure:

Use either 96-well plates, or Falcon 2025 tubes

 

Buffer:

·      1X PBS

·      1% FCS

·      0.02% azide

 

Cells:

·      Use 10⁵ - 10⁶ cells.

 

1.     Add equal volume of cells to positive and negative control groups as well as experimental samples.

2.     Add 1 ml buffer.

3.     Spin 3 minutes at 1000 rpm and 4° C.

4.     Pour off supernatant.

5.     Add first antibody to positive tube only at proper dilution. (100ul)

6.     Incubate for 30 minutes on ice.

7.     Wash and spin twice.

8.     Add second antibody to both tubes at proper dilution. (100ul)

9.     Incubate for 30 minutes on ice.

10.  Wash and spin.

Resuspend in 200ml of buffer.

 

 

Basic FACS for Mouse blood

1.     Add 35ml Heparin to microcentrifuge tubes.

2.     Add blood.

3.     Add 1ml ACK lysing buffer.

4.     Incubate 10 minutes, Room Temp.

5.     Spin 3-4 minutes at speed “4” on the microfuge.

6.     Wash in 1ml PBS-FACS buffer.

7.     Remove supernantant.

8.     Resuspend in 200ml PBS-FACS buffer.

9.     Transfer to plate

10.  Wash in 200 ml and spin 3 minutes at 1000 rpm.

11.  Continue with step number 5 from above.

 

Note: For tubes, staining volume is 100ml and washing volume is 1 ml. For

plates, staining volume is 100ml and washing volume is 200ml.