Lab Techniques (Research)

Hi there! Hope you guys are enjoying your work~

Right, I'll be focusing on the experiment that I did for my MP rather than SIP. That's
because the SIP job scope is mainly setting and clearing up after the
students' practicals. So that's why wemusn't abuse our position as students when we come back. It's all hard work being a TSO.

As most of you know, I'm working on the same project as Joan, just only looking at different components.

After 3 continuous weeks of gavaging the mice, it was time to sacrifice them. The mice were anaesthesized with Ketamine, a dissociative anaesthesia, and Pamlin, a sedative through Intraperitoneal (IP) injection.
How much to be injected was worked out by using this formula:
Dosage to be given = (Weight of mouse(kg) X Drug Dose)/Concentration

After they were all sedated, blood was collected by Cardiac Puncture and transferred to EDTA Tubes to prevent them from clotting.

The mice were then euthanized by cervical dislocation. The mouse was placed facing down, its neck was held down in place and the tail was pulled hard swiftly. This will then break the backbone of the mouse and sever the nerves connecting the backbone and the brain. This is one of many humane methods of euthanizing.


Diagram 1 shows the position on euthanizing the mouse using cervical dislocation.

The heart was checked to see if it was still beating before continuing on with dissection.
For dissection, a dissecting tray, a pair of forceps and scissors were prepared by swapping with 70% alcohol to disinfect the equipments. The mouse was placed onto the dissecting tray with its stomach facing up, a midline incision was made by using the forceps to grab the skin and the scissors to cut. The spleen was removed and placed into a tube containing 1X Phosphate Buffer Saline (PBS). The spleen is found at the left side of the mouse, at the posterior. The liver was removed and placed into 1X PBS with 0.16mg/ml of Heparin. The liver is directly below the lungs/ribcage.


Diagram 2 shows how the dissection was done and where the organs are found.

Thereafter, each mouse was placed into the biohazard bag and placed into the freezer for the storage of carcasses.

LDH Assay
First part:
The spleens were taken to perform the LDH Assay.
Working in a flowhood, each spleen was homogenized by scraping them against a sterile cell strainer into a suspension in 10ml of RPMI + 10%FBS medium.
Working in the dark, 5ml of Lymph M were pipetted into 12 15ml centrifuge tubes.
* Lymph M is a solution that works by density gradient. After centrifugation, different cell layers will be formed by their densities, including the lymphocyte layer.
5ml of the splenocyte suspensions were slowly aliquoted into the tubes using a glass pipette so as not to disturb the interface.
The tubes were then centrifued at 1500g, for 20 minutes at room temperature.
The yellowish lymphocyte layer was removed using a glass pipette into a new sterile 15ml centrifuge tube.


Diagram 3 shows the different layers after centrifugation.

The removing the lymphocyte layer, the lymphocytes were then washed 2 times by adding 10ml of medium and centrifuging at 800g, for 10 minutes at room temperature, decanting the medium then resuspending the cell pellet in another 10ml of medium.
After washing, 1ml of RMPI + 2%FBS medium was added into each tube and the cell pellets were resuspended.
Using a haemocytometer and tryphan blue, the lymphocytes were counted.
YAC-1 cells cultured were also counted using the above method.
Calculations were made to challenge the lymphocytes at different cell concentration, namely, 50X, 100X, and 150X.
A 96-well plate was used for the challenging of the cells.
From here onwards, RMPI + 2%FBS medium was used instead of RPMI + 10%FBS.
Blanks, High and low controls, effector controls and the test samples were done in triplicates.
The plate was then incubated in a 37oC incubator with 5% CO2 overnight.

Second part:
The next day, all the suspensions were pipetted into eppendorf tubes and centrifuged at 1500g for 5 minutes at 4oC to form the cell pellets.
Cell lysing solution was made, by aliquoting 100uL of Triton X into 10ml of RPMI + 2%FBS and vortexing the tube for Triton X to dissolve.
The high control supernatants were decanted and 200uL of the lysing solution was added to lyse the cells.
2 plates of 96-wells plates were used for the assay as each tube was done again in triplicates.
A 20X dilution was made to each tube by adding 95uL of DI water to 5uL of each cell supernatants into the new 96-wells plates.
The reaction mixutre was prepared by pipetting 1ml MilliQ water to the lyophilzate catalyst for 10 minutes and vortexing it after 10 minutes.
The dye was thawed in the water bath.
250ul catalyst was pipetted to 11.25ml of dye into 15ml centrifuge tube for 100 tests and the mixture was mixed by inverting up and down.
This reaction mixture was poured into a resevoir and 100uL of it was pipetted into each well using a multi-channel pipette.
The plates were then covered with aluminium foil and incubated for 20 minutes.
The stop buffer was prepared by pipetting 80ul DI water to 20ul 5M HCl to a dilution of 1N HCl
* Always add acid to water slowly!
After incubation, 50uL of stop buffer was pipetted into each well using a multi-channel pipette to stop the reaction.
Absorbance were then read at 490nm by using a spectrophotometer.
*Any bubbles formed will affect the results, thus, all bubbles must be removed before reading the plate.
Cytotoxicity was calculated using this formula:
((Effector-Target Mixture – Effector Control) – Low Control))/(High Control – Low Control)


Diagram 4 shows YAC-1 cells at 20X


Diagram 5 shows the lymphocytes at 10X


Diagram 6 shows the lymphocytes with YAC-1 cells at 10X

All diagrams and photos taken/done by me.

That's all!
Any questions, just ask me! =DD

Posted by:
Charmaine