Lymphocyte Culture

1. Glass vacutainer tubes containing heparin (Becton Dickinson, East Rutherford, NJ).

2. RPMI-1640 culture medium (RPMI-1640) (Gibco Invitrogen, Grand Island, NY) supplemented with 20% fetal bovine serum (FBS; Gibco Invitrogen). Stored at 4°C.

3. Gentamycin sulfate (Irvine Scientific, Santa Ana, CA). Added to RPMI-1640 at 10 mg/mL.

4. Sodium heparin (Elkins-Sinn, Cherry Hill, NJ). Stored at 4°C and added to RPMI-1640 at room temperature (1000 U/500 mL).

5. L-Glutamine (Gibco Invitrogen, Bethesda, MD). Stored at -20°C and added to RPMI-1640 at room temperature (4 mL/500 mL).

6. Phytohemagglutinin (PHA), lyophilized (Gibco Invitrogen): Reconstitute one bottle of PHA with 10 mL of distilled water. The reconstituted PHA should be stored at 4°C and prepared fresh every 30 d. It should be stored at 4°C and reconstituted at room temperature in distilled or deionized water.

7. KCl-Na citrate hypotonic solution (Fisher Scientific, Pittsburgh, PA): Add 0.560 g of KCl to 100 mL of distilled or deionized water to make a 0.075 M solution. Add 0.8 g of Na citrate to 100 mL of distilled or deionized water to make 0.8% Na citrate solution. Mix the two solutions just before use: 2:1, KCl:Na citrate. Store at room temperature and prepare a fresh mixture every 7 d.

8. Fixative solution: 3:1 methanol:glacial acetic acid.

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