Cannabinoid Drug Incubation and Preparation of Slides

The aim of our project was to determine the effects of cannabinoid drugs on HL-60 cells and whether they had the ability to induce apoptosis. To do this we initially chose 4 different cannabinoid drugs which were incubated with HL-60 cells for 2 hours. The drugs used were: AM630, AM281, Oleamide and 0-2545. These were chosen as they are a mixture of synthetic and endocannabinoids.

After counting the number of cells on the TC20 automated cell counter, we calculated and added amount of cells needed to have a concentration of 1×105 in 3ml of Roswell Park Memorial Institute (RPMI) cell culture medium. The drugs were added to individual wells in a tissue culture multiwell plate. A control was also set up which contained the same amount of the solvent used to dissolve the drugs dimethyl sulphoxide (DMSO), in the absence of any drugs.

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Corinne carefully resuspending the HL60 cells in DMSO.

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Lee-Anne apirating HL60 cells into culture multiwell plate.

The culture multiwell plate was then covered and placed in the cell culture incubator at 37°C and 5% COfor around 2 hours.

Cytoslides were labelled and placed correctly in cytofunnels and then into the Cytospin centrifuge, ensuring it was equally balanced. 0.5ml of each drug or DMSO incubated cells were then added into the cytofunnels, which were centrifuged at 2000 rpm for 10 minutes.

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After the Cytospin centrifuge finished its programme, the slides were taken out of the cytofunnels and chemically fixed onto the cytoslide surface in 100% cold methanol for 20 minutes. They were then left to air dry and placed in a -20 centigrade freezer before staining.

Glass slides were also prepared to carry out fluorescent staining. The cannabinoid drugs used for incubation were AM281, Oleamide and NADA. We also chose to use two of the original drugs that produced the most prominent results after observations carrying out cytological staining. As the cells were fairly sparse using 1×10cells/ml, we decided to increase the number of cells to 5×10cells/ml.

The cell samples used for fluorescent staining were incubated with the drugs for 2, 4 and 24 hours. At this time, the number of cells were also counted using the TC20 automated cell counter.

After each incubation, each sample was processed on the Cytospin centrifuge. The slides were fixed in methanol free formaldehyde for 20 minutes and left to air dry before being placed in a -20 centigrade freezer prior to staining.

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