Day 8

For today and tmr we would be under Dr. Wen Feng. We are back at the MSE. What we did today were tensile testing, cell seeding and DSC.

The schedule for the next two days. 

Tensile testing is used to test how much a material can stretch to. Researchers working in materials science use this test for a variety of reasons, such as to select the appropriate materials and quality control. From this test, we can also predict how the material would react when other types of forces are applied. The polymer we used was PCL. It was tough and was similar to the plastic we use to wrap our textbooks with (ignore if you don't know).

1. Punch (like hole puncher that kind of punch) a thin layer of polymer using a press, choosing the proper shape-circle and rectangle (it looks like an hour glass) shapes. The circle ones are not used in this procedure, it would function as a scaffold for cell seeding later. 
2. After measuring the thickness of the polymer using an electronic micro screw gauge, we used an universal testing machine to test the tensile strength of the polymer.
3. The machine uses the distance the polymer can stretch before breaking to calculate the tensile strength.

This is the PCL polymer that we used. 

The "puncher"

Press hard!

The rectangle shaped stamp would punch out something like this. This isn't the polymer, this is just aluminium foil.

This is the polymer, but apparently the stamp blade wasn't sharp enough. So we had to cut ourselves.

TA-DA! This will be used for tensile testing. 

Electronic micro screw gauge. Saves you all the trouble of reading the measurements. If all of us use this in schools, we would never have to learn how to read a micro screw gauge. 

The universal testing machine. Its huge. 

Just a frame to hold the polymer before attaching it to the machine. 

Stretching in progress.

PIAAK! (There wasn't any sound.) 

If you compare the two pictures, you will realise that the polymer in the first picture is slightly white in the middle, but not in the second one. This is because the polymer is elastic and would return to its original state after a while (It will still remain broken. It is not magic.)

The results! The sharp drop was when the polymer broke.

In cell seeding, we placed fibroblast that Dr Wen Feng showed us on Day 4 on different types of scaffold.

1. added 70% ethanol and left under UV light for half hour to fully sterilise the scaffold. The scaffold used were pieces cut our from those we made in electronspinning in Day 2 as well as the circular polymers we punctured earlier in the day.
2. After sterilisation, the scaffolds are washed in PBS thrice with half hour intervals
3. we viewed the fibroblast cells under the electron microscope
4. Trypsin is added to detach the cells from the plate surface and incubated at 37 degrees C for about 2mins.
5. Centrifuge to spin down the cells and pour away the supernatant.
6. Culture medium is added to the cells pipetted up and down to ensure a homogenous mixture.
7. A small sample was taken and diluted to get a 10x dilution and we counted the cells. It was tiring and difficult because there were about 100+ cells in each grid.
8. The remaining cells are placed on top of the scaffolds we prepared earlier and incubated at 37 degrees C overnight.

Stainless steel weights are added so that the scaffolds would not float. 

MILLIONS OF CELLS.

This is a disposable hemocytometer made out of plastic. The one we used with Ajay is reusable. If you want to know more about counting cells, check out our Day 4 post! 

We loaded the samples ourselves! 

Hard at work counting cells. Poor Alethea had to count in her head. She lost count many times.

Adding DMEM after the cells are added onto the scaffold. Dr Wen Feng explains that a smaller well tray is used because less solution will be required and hence saving resources. 

Differential scanning calorimetry (DSC) is a technique which it measures the difference in amount of heat required to increase the temperature between a reference and the sample. The reference is like a control, it is just the same container without any sample in it. The difference would allow us to find a few things like the melting point of the sample. Throughout the experiment, the temperature of the reference and the sample should be similar. But when the sample undergoes a change in state, it absorbs or release heat energy (depending on what is the change). Thus, the sample would require a different amount of energy as compared to the reference.

1. preparing the reference and sample. We only required 3mg so tiny tiny pieces of PCL was weighed using a electronic microgram weighing balance.
2. Sample was put in an aluminium pan and sealed with a press.
3. Loaded sample and reference into the machine and the procedure was designed before it was started. The machine would take slightly more than 2 hours to complete the cycle.

Electronic microgram weighing balance. 

The small little bits of PCL above the "s" and the "m" are all that we need.

We sealed the PCL inside these small little aluminium pans. Dr Wen Feng says that only a small amount is needed because sometimes the compound that are used are very expensive. 

The machine we used for DSC.

Different kinds of pans are used for different things. This is used to hold liquids.

Whereas this is used to hold solids. 

Loading in the reference and samples!

How to operate 101

This is process that we designed. Ramp means increase/decrease temperature. Isothermal means keep constant. 

Here is the results of our DSC. Actually we only got the results the next day because we ran out of time. The peaks on the graph show us the melting points and the freezing points of PCL. The peak at the top is the freezing point. While the peak at the bottom is the melting point. There are two peaks at the bottom, but the smaller peak is the correct melting point. The larger peak is inaccurate because it changes for each sample depending on various conditions such the structure of the sample. For example, if the PCL was pulled into fibres using electrospinning or casted into a  thin film, the larger peak would be different. However, the smaller peak would remain the same as long as it is the same material.

According to the schedule for today, we were supposed to visit the SBS Animal House. But due to time constraint, we couldn't go. ): Dr Wen Feng promised us to bring us there tomorrow, so all of us are looking forward to tomorrow! Stay tuned!

Day Seven

Continuing where we left off ytd, today we continued with western blotting as well as starting on flow cytometry.

For western blotting:

1) The overnight primary antibody incubated membrane blots were washed thrice with tris-buffered saline (TBS), containing 0.1% tween (TBST).
2) Secondary antibodies targeting the specific animal species of the antibody was added. For example: Goat anti-rabbit secondary antibodies were added to detect rabbit derived anti-human tubulin antibodies. While donkey anti-goat antibodies to detect goat derived anti-vimentin antibodies.
3) The cells undergo incubation for 1hour at room temperature.
4) After washing thoroughly with TBST again, the membrance blots were dried in a 37⁰C incubator.
6) We used an scanner to scan the dried membrane.

For flow cytometry, we used the cells we had from ytd. Those with Palitaxel and those without.

1. We washed the cells with PBS for about 3 times
2. Trypsin is added to detach the cells from the plate surface. Weak trypsin is used for MB 231, unlike for RT3 and MCF-7. This is because MB 231 cells are less attached to the plate surface so less trypsin is required. 
3. The excess trypsin is neutralised with PBS or 0.1% fetal bovine serum (FBS). 
4. Centrifuge to spin down the cells, and discard the trypsin carefully.
5. 70% ethanol is added for cell fixation, which preserves the cells in it's life-like state although the cells are already dead. 
6. Wash with PBS with 0.5% BSA. This prevents the clumping of cells, ensuring even dispersing.
7. Hydrochloric acid is added to denature the DNA by disrupting the hydrogen bonding of the DNA duplex, hence unwinding the DNA helix to expose the thymidine analog (BrdU).
8. Cells are washed with PBS with 0.5% BSA before the addition of FITC conjugated anti-BrdU antibodies. FITC is a green fluorescence dye. 
9. Incubate for 1 h at 37⁰C.
10. Propidium iodide (PI), a DNA binding dye, is added to the cells before subjecting to flow cytometry analysis. 

Ivan used this motor to suck up the remaining cell culture medium. COOL.

This box is used to store the membrane with the antibodies during incubation. The antibodies are light-sensitive so they cannot be exposed to light for long.

Having fun pouring the supernatant away after each centrifugation. 

Propidium iodide. It looks like a carrot. 

The machine we used for flow cytometry

Ideal BrdU results. Ours didn't come our ideally. :(

Scanner for western blotting

This is a guide for the protein ladder. The different colours and height represent the varying mass.

Western blotting results for tubulin

Western blotting results for vimentin

We had a great time with Ivan. Thanks, Ivan! (BTW he sat down, so he doesn't look very tall)

So true.

KEHEHEH.

This are some of the other guys who work in the same lab as Ivan. We had a lot of fun in SBS partly because of them. As quoted from Mingjie, they are a "fun-loving bunch". XD

Mingjie and Ivan gave us this one-hour talk about how research career is like. The moral of the story is you must have passion in science if you want to join the research career. Mingjie was discouraging us to join research and Ivan was encouraging us. HEHE. But all of them have a passion for science and that is what matters. 

IVAN IS TALL!!!!!!!!

Its been a while since we posted our own individual views. But I really want to share my experience at SBS. So as explained above, the labs and office in SBS are next to each other. Personally, I prefer SBS to MSE, mainly because of its environment. In the SBS lab, I could feel that there people working inside are pretty close to each other (let's hope I am not wrong). They would help each other to do small small things, like preparing solutions or even buying food. There is friendliness and warmth in that lab and I hope I would get to work in such an environment in the future. I hope I can return there someday again. Hehe.

Mingjie and Ivan also talked to us about how research is like and the pros and cons of it. After talking to them, I could feel the passion they had for science and research and I really admire them for that. I also felt that the one hour spent talking to them was worth it, even though I was starving already. What they said are really their own experiences and they gave me a realistic view on how research is like. It is better than all those talks or open house events. Although I was slightly put off from research from Mingjie's words, I still love science and have no other intention of deviating from it. 

Karmen

Hello! It's time for our individual reflections! Actually, I also share the same sentiments as Karmen. I really like the SBS's labs and the people in there. They helped each other a lot, by helping prepare the other person's solutions (if he really needs it) and joked a lot with each other. It was really a lively place! I also learnt a lot about the different methods used in molecular biology. It was related to the work that we learnt in school. So, I was able to understand the things that Ivan was saying. In the lab, there were really a lot of machines to use and know. They have to know the different computer programmes which are associated with the different machines. It was really amazing! Anyways, the one hour lecture from Mingjie and Ivan was quite inspiring to me, as I had never seen people who had that much passion for their work and projects. They really love the things that they do, even though it may be tough most of the times. Looking at them speak about research with so much passion, I was awed and felt a bit guilty. They not only talk about research, but they also talk about the different areas (mostly, of the cons). It made me think of my future career path and what I would really like to see myself doing in 20 years' time. To know that they are driven mainly by their passion and interest in the fields, it made me feel that the people who are out in the workforce are working on the things that they love, despite the lack of sleep and the many things that they had to read on! So, I really want to thank Ivan and Mingjie for teaching me that valuable lesson.

Alethea

Hi! This is Yuh Harn. i fully agree with what Karmen and Alethea said in their comments. Indeed the environment at SBS is more fun-loving and more informal than in MSE. Random jokes and a helping hand from time to time would be really nice. But for me, perhaps it's just my personality, but i would prefer working at MSE, with a more distraction-free environment when working. A little chat and laughter here and there but when working i would prefer full concentration. Everyone has different preferances, so this is just mine. And it has been great talking to Ivan and MJ about career choices, having to choose uni courses within the next few months can feel stressful and i'm glad to have had a 'prep talk' by them.

Yuh Harn

And that marks the end of our time at SBS. Not only did we had a fun time, we also made new friends with the guys in the lab. They have also became our supporters! We thank them very much for their support and patience with us for the past two days! We wish them the best from here on. 

We will be working with Dr Wen Feng tomorrow back at MSE. Cheers!