Preparation of single-cell suspensions of bone marrow cells

Summary

This experiment is based on "Color Atlas of Practical Flow Cytometry", edited by Shukui Wang and Zhenying Zhou.

Operation method

Preparation of single-cell suspensions of bone marrow cells

Materials and Instruments

Bone marrow fluid
Heparin anticoagulant PBS

Move

1. Aseptically aspirate 0.5 ml of bone marrow fluid.

2. Place the bone marrow specimen into 1 ml of PBS containing 1000 U/ml heparin anticoagulant.

3. Dilute to 10 mI with additional PBS.

4. Pipette 5 ml of diluted bone marrow into 4 ml of human lymphocyte isolate.

5. Under the above conditions, the bone marrow nucleated cells are layered at the interface between the PBS Human Lymphocyte Isolate.

6. Aspirate the layer of nucleated cells into 10 ml of PBS and mix well.

7. Centrifuge at 1000 r/min for 5 min and discard the supernatant; collect the bone marrow cells and add fixative or set aside in a freezer.

Caveat

1. When aspirating bone marrow fluid, first moisten the syringe tip, needle barrel and needle plug with heparin, so that the force of aspiration is moderate.

2. When aspirating the lymphocyte layer, try to absorb as little separating fluid as possible, and the volume of aspiration should be kept at about 300 ul, so as to help wash away the excess separating fluid.

3. The method of dissolving erythrocytes: add an equal amount of distilled water to the precipitate, shake gently for a few moments, see the appearance of pink color, immediately add a large amount of saline, mix, centrifugation, and remove the supernatant can be.

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