This lab was derived from: Animal Cell Culture - A Guide to Basic Techniques (5th Edition)

Operation method

Scheme 27.2 Stimulation of lymphocytes with PHA

Materials and Instruments

Culture medium with 10% FBS or autologous serum Phytohaemagglutinin Colchicine Amide
KCl
Test tubes or plain containers Slides for microscopic use

Move

1. Take the cells washed in Step 7 of Scheme 27.1 and incubate them in HEPES or _CO2 buffered DMEM, CMRL 1066 or RPMI 1640 with 10 % autologous serum or FBS at a cell density of 2 × ¹⁰⁶ cells/ml and at a depth of 1.5 to 2.0 cm.

2. Add PHA at a final concentration of 5 μg/ml and stimulate mitosis for 24~72 h.

3. Cells were collected at 24, 36, 48, 60 and 72 h, and smears or slides were prepared by centrifugation to determine the optimal incubation time (i.e., maximum division index).

4. add colchicine amide (prepared with BSS) at a final concentration of 0.001 μg/ml and treat for 2 h. The peak of mitosis occurs earlier than that observed in step 3 [ Berger, 1979].

5. After colchicine amide treatment, the cells were centrifuged. Chromosomes were then prepared by suspending the cells with 0.075 mol/L KCl to allow hypotonic swelling of the cells (see Scheme 16. 7 and Scheme 27. 5).

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