Author: SCJ. Date: 1/2/96

Standard Operating Procedure:

⁵¹Chromium Release Assay

Hazards:

Radioactive ⁵¹Chromium

Refer to the University of Minnesota Radiation Protection Guide for information on the proper use, shielding, and exposure limits.

 

All radioactive work must be done in approved and posted radioactive use areas.

Protection:

Lab Coat

Gloves

Dosimeter ring and badge

Lead Shielding

Geiger Counter for monitoring radiation exposure

Absorbent paper or blue pad

Waste:

Solid waste should be disposed of in container for short half-life isotopes located in 7-159. Included in solid waste is bench paper, pipette tips, dried plates, kimble tubes, and similar materials.

 

Liquid waste should be stored in vacuum trap located in 7-155. Trap level should be monitored regularly.

Spill Clean-Up:

Follow procedure outlined in the University of Minnesota Radiation Protection Guide page XII-1.

 

Decontaminate area with Lift-Away which is located under the main sink in 7-159.

Target Cells

 

1.     Count cells and spin down appropriate number to approximately 10⁶ cells/200 ml RP-10 in microcentrifuge tube.

2.     Add 100 mCu ⁵¹Cr/10⁶ cells. Increase volume of ⁵¹Cr as it decays.

3.     Record date, amount of isotope used, activity used, and amount remaining on inventory forms. Complete Radioactive Waste Inventory forms for both liquid and solid waste.

4.     Add peptide if appropriate. (i.e. antagonism assay)

5.     Incubate cells for 1 hour at 37° in appropriately labeled incubator.

6.     Wash cells 3X in RP-10. Aspirate liquid waste into reservoir in 7-155.

7.     Resuspend in appropriate volume RP-10 for assay. Target cells are typically used at 10⁴ per well and 50 ml per well.

8.     Treat all pipettes and other materials that contact radiolabeled cells as radioactive

            and discard appropriately.

 

 

Effector Cells

 

1.     Count appropriate effector cell(s).

2.     Resuspend correct number in RP-10. Effector cells are typically used at 3x10⁴ per well in 100 ml per well.

 

 

Peptide(s)

 

1.      Peptides are typically diluted in 50 ml PBS per well.

2.      If serially diluting, be sure to change yellow tips between wells.

 

Controls

Spontaneous Release:

1.      50 ml Target cells

2.      150 ml PBS

3.      Always done in triplicate.

 

Detergent Release:

1.      50 ml Target cells

2.      100 ml Triton-X detergent

3.      50 ml PBS

4.      Always done in triplicate