Protocol for the Preparation of Human Peripheral Blood Chromosomes

Human peripheral blood small lymphocytes, which are usually in the G1 (or G0) phase, do not normally divide. If phytohemagglutinin (PHA) is added to the culture medium, such small lymphocytes can be stimulated to transform into lymphoblasts and enter mitosis. After short-term culture, a large number of mitotic cells can be obtained by colchicine treatment, hypotonicity, and fixation. 1 ml of human peripheral blood generally contains about 1×10⁶ to 3×10⁶ small lymphocytes, which is sufficient for chromosome specimen preparation and analysis.

• Sterilize the skin with alcohol, and collect blood from the elbow vein 0.3-0.5 ml. Immediately pass the syringe needle directly through the rubber stopper of the culture bottle, inject 30-40 drops of whole blood into 10 ml of culture medium, shake gently, and set it in a 37°C constant temperature box for incubation.
• Cultivate for 68 hours. During the incubation period, the cells are shaken gently periodically to make them full contact with the culture medium.
• 2-4 hours before termination of culture, colchicine (final concentration of 0.07 μg/ml) is added to the culture medium.
• Transfer all the cultures into clean centrifuge tubes and centrifuge at 1000 rpm for 8-10 minutes, discard the supernatant.
• Add 8 ml of pre-warmed 37°C hypotonic solution to the graduated centrifuge tube and mix well with a dropper. Place in a 37°C constant temperature water bath for 15-25 minutes.
• After hypotonic, add 0.5 ml of fixative, mix gently, and centrifuge at 1000 rpm for 8-10 minutes.
• Discard the supernatant, add 5 ml of fixative, mix gently, and leave for 20 min. Centrifuge at 1000 rpm and discard the supernatant. Repeat the fixation three times.
• After discarding the supernatant, depending on the number of cells, add the appropriate amount of fixative to make cell suspension.
• Aspirate the cell suspension from a height of 10-20 cm, drop it onto a dry and clean slide, blow gently, and air dry.
• 1:10 Giemsa staining for 5-10 minutes, fine water to wash away excess staining solution, air dry.
• Look for well-dispersed, moderately stained split phases under low magnification, observe chromosome morphology under an oil microscope, and count.

Creative Bioarray Relevant Recommendations

• Peripheral blood mononuclear cells (PBMCs) are cells with a single, round nucleus and are collected from the peripheral or circulating blood by density centrifugation with Ficoll, a polysaccharide. PBMCs include dendritic cells, monocytes, and lymphocytes (T, B, and NK cells).
• Apart from that, Creative Bioarray offers different ranges of human blood samples, such as whole blood, blood serum, plasma, and coagulation factors.

Colchicine needs to be sterilized and dispensed. Carnoy fixative and Giemsa stain, need to be prepared at the time of use.

Peripheral Blood Mononuclear Cells

Blood

For research use only. Not for any other purpose.