Preperation of Reagents

DOE-SPEC-1142-2001

5.1.8. Density gradient solution for blood mononuclear cell separation (i.e., Ficoll-

Hypaque, Histopaque, etc.). Once opened, store at 4C and warm to room

temperature before use. (For Blood BeLPT only.)

5.2.

PREPARATION OF REAGENTS

5.2.1. Complete growth medium should be prepared using sterile technique to a final

concentration of 10% human AB serum, 1% L-glutamine, and 1% penicillin-

streptomycin in RPMI-1640 (e.g., to 1 x 500 ml bottle RPMI-1640 add 55 ml

human AB serum, 5.5 ml L-glutamine [200 mM] and 5.5 ml penicillin-

streptomycin [100 x]). Prepare the quantity that will be used in a week. Store

at 4C.

5.2.2. Tritiated Thymidine (thymidine, [methyl-3H])). Note: Since tritiated

thymidine is a radioactive material, appropriate safety precautions must be

applied to prevent spills, to properly label, and to dispose of contaminated

pipettes, vials, microtiter plates and other items in contact with this substance

as well as all liquid wastes. Laboratory-specific requirements for technician

training and waste disposal should be developed in consultation with the

appropriate radiation safety officer. Prepare a new lot number of tritiated

thymidine in the following manner: dilute 1 mCi/ml of tritiated thymidine

with the medium prepared in step 5.2.1 (e.g., 1 ml tritiated thymidine + 19 ml

Medium).

5.2.3. Beryllium Sulfate (BeSO4), store at room temperature. Same procedure for

other salts of beryllium if used. Prepare BeSO4 dilutions fresh for each assay,

using the 0.2 Molar (M) stock solution. Sterilize the solutions by filtration.

5.2.3.1.

Make a 1:10 dilution of (BeSO4) solution with phosphate buffered

saline solution, Ca++ and Mg++ free.

5.2.3.2.

Continue making serial dilutions with Complete Medium to create

three dilutions of 2 M BeSO4, 2 0M BeSO4, and 200 M

BeSO4. When added to wells, this will give a range of

concentrations of beryllium from 1, 10, and 100 M BeSO4.

5.3.

QUALITY CONTROL

5.3.1. New lots of AB serum are tested for ability to support a response to beryllium.

Specifically, the serum should produce stimulation indexes of approximately 1

in beryllium-challenged wells for non-sensitized subjects. Also, it should

demonstrate low toxicity or cell-killing [i.e., not more than one standardized

Ln(SI) (See B.6) should be less than 3.0] in the presence of beryllium. The

serum should NOT stimulate excessive lymphocyte proliferation in control

wells.