PreparationOfPeripheralBloodCellsForChromosomeAnalysis

实验概要

Lymphocytes  are differentiated cells which normally do not undergo subsequent cell  divisions. By culturing lymphocytes in the presence of a mitogen  (KARYOMAX® Phytohemagglutinin (M-Form) (PHA), Cat. No. 10576), they are  stimulated to replicate their DNA and enter into mitosis. After an  optimum time of the cells being cultured (46 h for a newborn and 68 h  for an adult), a mitotic inhibitor, KARYOMAX COLCEMID® Solution (Cat.  No. 15210 or 15212), is added to the lymphocyte culture for 20 min.

The  addition of COLCEMID to dividing cells acts to prevent the synthesis of  spindle fibers and, therefore, to stop mitosis in metaphase. Metaphase  is the optimum phase of mitosis for microscopically visualizing the  chromosomes. By submitting cells to a hypotonic solution and a series of  fixation steps, metaphase chromosomes can be microscopicallyobserved  and analyzed.

As  a quality control measure, each lot of Phytohemagglutinin is tested as a  chromosome reagent for the examination of metaphase spreads used for  cytogenetic studies. This reagent is evaluated by supplementation to an  approved, non-phytohemagglutinin containing chromosome medium. These  samples are then supplemented with freshly collected human peripheral  blood and have been found to be acceptable in their ability to produce  blastogenesis with human lymphocytes when compared to a previously  tested control.

主要试剂

Phytohemagglutinin (M Form) (PHA), Liquid [Details:Life Technologies Cat# 10576-015]

PB-MAX™ Karotyping Medium (1X), Liquid [Details:Life Technologies Cat#12557-013]

KaryoMAX® Colcemid™ Solution, Liquid (10 µg/ml), in HBSS [Details:Life Technologies Cat#15210-040]

KaryoMAX® Colcemid™ Solution, Liquid (10 µg/ml), in PBS [Details:Life Technologies Cat#15212-012]

实验步骤

1.        The required volume of peripheral blood is collected aseptically in a sodium heparinized vacutainer tube or syringe.

2.         Add 10 ml of either PB-MAX Karyotyping Medium or KARYOMAX Peripheral  Blood Karyotyping Medium to each sterile T-25 flask to be set up for the  assay.

3.        Add 0.75ml blood to each tube.

4.        Incubate flask in CO2 incubator for 48 to 68 h with caps loose.

5.        Add 0.05-0.1 ug/ml COLCEMID to each flask for a 15 minute incubation.

6.        After 15 min, transfer flask contents to a 15 ml centrifuge tube and spin down at 1,200 rpm for 5 min.

7.        Remove supernatant and resuspend pellet.

8.         Add 10 ml of 0.068 M KCl to pellet and gently mix. Allow to sit at room  temperature for 15 min. Add 0.5 ml of fixative (three parts absolute  methanol to none part glacial acetic acid). Gently mix with pipette.

9.         Centrifuge for 5 min at 1,200 rpm. Aspirate off supernatant. Add 10 ml  of fixative, mix, and let sit at room temperature for 10 min.

10.    Repeat centrifugation step. Add 5 ml of fixative, mix, and let sit for 10 min atroom temperature.

11.    Centrifuge and aspirate off supernatant. Add 5 ml of fixative and incubate for 10min at 4°C.