Bone Marrow Harvest

Hello everyone ! I’m Justin here again. Sorry for my super late posting. Hope all of you are enjoying your SIP so far =) Today I’ll talk about the harvesting process in more detail, covering the protocol, as well as some stuff to note.

As mentioned before in my previous post, harvesting is the processing step to obtain metaphases for analysis. The 3steps involved are mitotic arrest, hypotonic treatment, and fixation.

The cell cycle is arrested at metaphase by colcemid, which depolymerises tubulin, thus preventing spindle fiber formation. Although an increase in duration of exposure to colcemid causes more metaphases to be collected, a prolonged exposure to colcemid also causes chromosome contraction as an effect. This would result in shortened chromosomes, which are not suitable for analysis. Hence, the duration of colcemid exposure should be limited.

Hypotonic treatment causes water to enter the cell. 0.075M of potassium chloride is used. Cells would swell and increase in volume, thus causing chromosomes to spread out. The incubation at 37ºC will speed up the process. The duration of hypotonic treatment has to be controlled, as over-treatment will cause excess spreading of chromosomes and cell lysis.

The final step is fixation, using modified Carnoy’s fixative, which consists of methanol to acetic acid in the ratio of 3:1. The fixative has to be prepared fresh and refrigerated. This is because over time the fixative would absorb water from the surroundings, thus causing the fixative to lose its properties. Fixation hardens cells and makes them resistant to changes. Changing of the fixative (through centrifugation and removing the supernatant) also lyses red blood cells, providing a clear suspension.

In this protocol, the colcemid solution and potassium chloride are incorporated into 1 solution, known as the harvest media. 0.08µg/ml of colcemid is used. Cells are exposed to the harvest media for a duration of 30 minutes.

Method:
1.Obtain the cultures from the incubator (after their respective durations of incubation- recall first post on culture setup) and transfer the culture contents into a centrifuge tube. (This is done because the culture flasks cannot fit into the centrifuge) Centrifuge at 1500rpm for 10 minutes.

2.Remove the supernatant and add 10 ml of harvest medium to the cell pellet. (The harvest medium contains the colcemid and the hypotonic solution)

3.Incubate in a 37ºC incubator for 30 minutes.

4.After which, remove from the incubator and perform a pre-fix step by adding 2ml of fresh cold fixative. (The purpose of the pre-fix step is because cells are fragile after the 30 minutes exposure to the hypotonic solution. The fixative will harden the cells and prevent them from being destroyed by the force of centrifugation) Ensure that cell clumps are well dislodged by mixing well.

5.Centrifuge at 1200 rpm for 10 minutes.

6.Remove the supernatant and resuspend the cell pellet in 6 ml of cold fixative. (This is the fixation step) Ensure that cell clumps are well dislodged by mixing well.

7.Centrifuge at 1200 rpm for 10 minutes.

8.Remove the supernatant and resuspend the pellet in 4ml of cold fixative. (this is to change the fixative) If supernatant is still coloured, change the fixative (through centrifugation, removing the supernatant and re-suspending the pellet in 4ml of cold fixative) until it becomes clear.

9.Place the tubes in the refrigerator. (the next process would be slide making)

Per culture- 10 ml of harvest media
12 ml of fixative (2ml pre-fixation+6ml fixation+ 4ml changing of fixative)

That’s all for now. Take care everyone and enjoy SIP !

Ng Tze Yang Justin
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