Books: I found some great books :D

Last weekend, I went to Koromangalu (um... think that's how its spelled) mall in Banglore. There was a pretty big book store so I indulged myself in the science section as how any dutiful geek should do xD

Well I found some great books and here they are

new theories of everything by John D. Barrow
ISBN 978-0-19-954817-0

I've only finished the first couple of chapters of this book, but I can definitely tell that its a well written book.

John D. Barrow talks about the latest concepts in theoretical physics and mathematics that are geared towards finding the "theory--or theories-- of everything", sometimes referred to as M-theory.

Barrow talks a lot about the nature of the theory of everything, and throws many questions towards the reader than proposes his view on those questions. Questions like: What would a theory of everything look like? Would there be any initial conditions for such theory, and if there were, what would they be?

Most of the concepts he mentions are indeed very complex, but he is very adept at mentioning just enough about them so that even a layman could grasp the general overview of the concept. He also explains them quite well.

I'm eager to finish this book right now, but I'm saving it for my flight back to Cornell, cuz its gonna be a LOOOOOONG flight *sigh*


Second book I'll introduce today is titled:
How the Mind Works by Steven Pinker
ISBN 978-0-14-2-4491-5

Now I'm also saving this book for the flight, but I read one of the chapters "The Mind's Eye", as I'm very interested about how visual information is processed, also because I have some background in that area.

It was enthralled by the book instantly :D This book also harbors various concepts that are not recognized by laymen, but "Steven Pinker has a remarkable capacity to explain difficult ideas." (The Times)


It seems to me that both of the books promise a rewarding intellectual journey. I'll enjoy them to the full extent on my flight :D

Basic Lab Procedures: Freezing and Thawing cells into/from cryowells

Oftentimes in lab, situation arises when you need to store cells for future use. It is possible to store the cells without freezing them, but has many disadvantages like having to change the media regularly and the increase in passage of the cell or the like. So since the technology became available, freezing and thawing cells have been part of a very routine procedures in bio-tech labs.

Now what do we need for freezing and thawing the cells?

For thawing:
not much really... XD just your body heat will suffice.
but the chances are that you would want to plate the cells on to a T25 for something, so for that you would need
-a plate (t25)
-some media
-some tubes like 15ml falcon
and basic lab machineries like pipets, hood, and centrifuge.

For Freezing:
Need a little bit for this one :D
you would need
-trypsin (generally 0.25%)
-media
to harvest the cells from a plate and
-freezing media ( 10% DMSO, 90% FBS )
-cryowell
-cryo tub
and fridge/freezer, and liquid nitrogen freezer.

Now the How To part.

I'll start with freezing the cells.

-First, get the cells you want to freeze from the incubator.
-trysonize it to get the cells off the plate.
-once the cells are afloat, neutralize the trypsin with some media.
-centrifuge the tube(with cell/trypsin/media) at 1800 rpm for 5 min.
-pour out the liquid, while being careful not to disturb the pellet of cells.
-disturb the pellet and add freezing media. (add 1ml for each cryowell you'll be storing the cells, the number of cryowells would depend on how many cells are being frozen)
-mix the solution thoroughly to get a good suspension than add 1ml of the mix to each cryowell. (its specified as 1ml because the cryowells I've used have the capacity of 1ml, there are wells with different capacities, so note this.)
-put the cryowells in the cryo tub filled with isopropyl, and put the tub in -20'C
-after 30min move the tub to -80'C freezer.
-the day after move the crywells to the liquid nitrogen freezer (which is at -196'C)

NOTE: The cells are moved from freezer to freezer because if the cells freeze too fast the crystals that form will be sharp, causing the cells to rupture or lise. DMSO is included in the media to prevent this; however, the cells still have to be frozen slowly. For the same reason, the cryowells are placed in the alcohol tub. isopropyl has a property of freezing very slowly and steadily.

Now to the thawing part.
This is much easier :D
-retrieve the cells from the niquid nitrogen (be careful not to get burned by liquid nitrogen)
-thaw the cells by leaving the crywells in the water bath for a short while (or just hold the well)
-well there is still a small ice formation left, take the crywells out of the water bath and move them from the wells to a 15ml falcon tube (or any other centrifugable tube).
-Add few mls of media to the tube, and spin in at 1800 rpm for 5 min.
-discard the liquid, disturb the pellet and add appropriate amount of media to be used to plate the cells.

DONE :D

I'll be sure to get some pictures for this, I forgot D:

Internship Aug 4 2009

I know have 2 weeks left at MIRM and as i look back I realize how time flies. I've learned many things at the same time i haven't really learned anything. I mean i've learned some procedures and processes that would be used in culture work in any biological research lab. I would imagine that this knowledge would be useful in the future since i'm planning a career in bio-medical engineering. However, I feel that i didn't really take advantage of all the opportunities to learn things that I could only learn at this kind of lab (stemcell research lab). Although, having said that I don't think i would have been able to take in much information since I've know just finished my first year of my undergrad in Cornell. But there is a definite difference between "I tried, but couldn't get much out of it" and "I knew i couldn't get much out of it, so I didn't try". I would prefer myself in the first situation, as I've recently realized that attitude is more important than it seemed to me. So even though there is only two weeks left, I started today to ask questions about research goals and agendas, and grilled them for 'why's and 'how's about their research. They have explained some of their agenda to me in the very beginning, but I really wasn't able to understand much of it. Not just because of the foreign materials, but also because of their accent. Although Indian accent is still a bit of a problem, right now i feel much more comfortable with them :D. Even the materials seemed a little friendly.
I would love to write down the main research goal for the team, but they've asked me to keep the details to myself as they'll be using the information on their paper. (Dr. Jyothi's paper)
So here's a very short synopsis about it.
Right now there is a lot of research about stem cells are being done, but most of them are experiments on isolated settings. Realizing that cell's reactions can and will change in in vitro settings, our team focused the study on cell's response to cytokines released with inflammatory response.
This is the very basic gist of it.

Anyways Moving on.

Right now there is a crisis at MIRM because of some malfunction with their UPS.
If you didn't know already, India doesn't have a reliable, continuous power source like America. Banglore is a very big city so the power is more reliable than most part of India but with machineries that need constant power like incubators, people need to be sure that they will have continuous power. So they purchase UPS (Uninterruptible Power Supply) for this purpose. But apparantly the UPS at MIRM is malfunctioning, causing it to lost power once in a while. It loses power for only few minutes at a time, but every time the power goes out, all the incubators are turned off. this is casuing the cells to be stressed and is bothering the researchers a bit. There is a bigger problem tho, if the power goes out, when they are running samples on PCR or real-time PCR machines, the experiments get screwed up. This whole situation made me appreciate the constant power i had in America and in Korea.

Stem Cell Concepts: immunofluorescence (IF)

I've been quite lazy with these posts, and now there's a lot of pictures and ideas just tucked away somewhere and in my computer. I have 21days before i go back to Cornell, lets see how many i get up before i go :D










The concept i will introduce today is Immunofluorescence (IF).

Basically, IF is a labeling or tagging of specific sub cellular structures with fluorescence.
It is called "immuno-" fluorescence because it tags antibodies and antigens which is part of the immune system. I am not too knowledgeable about this, though, so i don't think i should try to explain how IF works.
I do know, however, why IF is used.

The reason why IF was developed, and all the other methods to 'tag' molecules, is very much related to the properties of light microscopy. Well, more like limitations of light microscopy. It is a general knowledge that cells or sub cellular under microscope do not have much color. The structures do not have much color to begin with and since the cells are often so thin they do not retain much color. To overcome this, many scientists have devised some methods to stain the cells so that it would be easier to make out the structures. 'Tagging' can be understood as more advanced form of staining. Researchers can now 'tag' specific types of structures with different type of tags to help that study their specimens. One of the shortcomings of staining was that it often requires the cells to be dead and treated, but tagging can be done to living cells, which allows the tracking of specific molecules or structures within the cells. This is rather significant because one could expose cells to different conditions and follow the tagged structures within the cells.

btw my explanations are rather amateur, and i apologize for that but stay with me, i still have some more to say :D

Another advantage to tagging is the specificity of the tagging, which is generally much more than what staining to offer. If a stain could stain an entire cell blue, tagging can tag just the DNA in the cell. (There might be a stain that could stain just the DNA, but i got my point across right?) And probably more significant than the other advantage is that the tagging can be read by computer very efficiently, while stained objects can not be read so efficiently. Tagging is done often with fluorescence, but this fluorescence cannot be seen by normally, it has to be first excited by laser than go through a filter before it could be seen. This information could be read by a sensor or a receiver which could be stored as a data of only the tagged molecules. Whilst if this was attempted with a normal stain on light microscopy would be hugely inefficient. Also staining is often only done once, but tagging can be done multiple times. If the staining is done multiple times the color of the stained area might change, but in tagging it won't happen because each fluorescence has to go through a different filter before it could be seen.

I'll just show you what i mean
these are pictures of Hela cells that have been tagged with DAPI and FITC. The second picture is the cell seen through the FITC filter and the third picture is seen through the DAPI filter.
The first picture is the composite of both pictures done on computer.




As you can see. DAPI stains the nucleic area while FITC stains the entire cell apart from the nucleic area. If you look closely, the area where neucleous would be in the second picture is a little less green.





I'll add a bit more explanation later, cuz its getting late right now, just some more pictures as of now.
















These are Bone Marrow cells I believe

Stem Cell Concepts: intro & What is Stem Cell?

Here's is another series of posts I am starting about random concepts about Stem Cells.
I will be writing about different kinds of Stem Cells, and about the concepts behind some of the conventional procedures like PCRs and molecular tagging and such.

I found it appropriate that I should start this series by clarifying what Stem Cell really is. :D

What is Stem Cell?
There are some misconception among popular culture that stem cells are type of cells that can give rise to all the cells in human body. Most of them often believe also that there is just one kind of stem cells. This is not entirely wrong, as there is one type of stem cell that give rise to all kinds of cells in human (well not necessarily just humans) body, the fertilized egg :D

There are two main ways in which the stem cells are categorized. They are categorized by their origin, (What I mean by the 'origin' is simply where the cells are found. For example, Bone Marrow Mesenchymal Stem Cells (BMMSC), are extracted from one's bone marrow.)
or their potency.
Potency specifies the differentiation potential (the potential to differentiate into different cell types) of the stem cell.[1] Which means that different stem cells can only differentiate into certain kinds or number of daughter cells. There are a lot of research done on the matter of cells' potency, so it is not absolute, but I think this definition will suffice here.

Here is the list of different types of Stem Cell potency:
Totipotent: Totipotent cells are fertilized eggs are cells within couple divisions since the fertilization. They can differentiate into both embryonic and extraembryonic cell types. This is what is different between totipotent and pluripotent stem cells. Embroyonic cell types are the ones that differentiate into one's body and extraembryonic cell types are the ones that differentiate into cells that support the bady in the womb.

Pluripotent: Pluripotent cells are decendents of totipotent cells and can differentiate into nearly all cell types, such as Embrynoic Stem Cells (ES cells) which can differentiate into all embryonic cell types. I will write more in depth about ES cells in the future

Multipotent: Multipotent stem cells can differentiate into few different cell types, but only those of a closely related family of cells. Much of the Adult stem cells are multipotent. Example of mulipotent cellss are BMMSC and WJ cells.

Oligopotent: stem cells can differentiate into only a few cells, such as lymphoid or myeloid stem cells. (from wikipedia)

Unipotent: cells can produce only one cell type, their own, but have the property of self-renewal which distinguishes them from non-stem cells (e.g. muscle stem cells). (from wikipedia)

Didn't learn about Oligopotent or Unipotent cells at my internship yet, so I paid wikipedia a visit ;)

If you didn't know already, Stem Cell research is one of the hottest field in Bio-medial research because it has a great number of possible usage and potential. It has been proven, though, that it is not easy to control stem cells as we want to, but number of stemcell treatments already exist and with time, stem cell will change the medical field greatly, as invention of medical imaging once did.




[1] - Hans R. Schöler (2007). "The Potential of Stem Cells: An Inventory". in Nikolaus Knoepffler, Dagmar Schipanski, and Stefan Lorenz Sorgner. Humanbiotechnology as Social Challenge. Ashgate Publishing, Ltd. pp. 28. ISBN 0754657558.

Basic Lab Procedures: Intro and Medium Preparation

I'll be uploading various procedures that I encounter during my internship for future reference and for anyone interested. This is the first of its series. I cannot guarantee that these posts will be in logical order, but these procedures are all very basic so the order might not be a big issue.

Medium Preparation

Culture Medium is a liquid or solidified nutrient material suitable for the cultivation of microorganisms

Different cells or bacteria need different kinds of media, and there are certain guidelines that different media should follow. I have not learned so much about them yet, so I will later post another, more in-depth post about media.

For now I will focus on the most basic kind of media used in MIRM lab, the Knock Out Media.

I can't really say this is much of a tutorial or anything, but its the first one, so i see no harm ;)

What You Need: (to prepare 50ml of KO media)

5ml of FBS
500ul of glutaMAX
250 ul of penstrep
45ml of Knock Out Media

In my lab, you can find them here :D
in the 4 'C fridge in the culture room.








Adding 5ml of FBS makes it a 10% FBS KO solution be sure to write that one the bottle.
Oh, and here's what you need to write on the bottle:
KO media - the name of the solution
+10% FBS - what is in the solution
+glutaMAX
+penstrep
0x/0x/0x -the date it was prepared
ASC - name of the group

Now here's a brief explanation of what FBS, glutamax and penstrep are:

FBS- FBS stands for Fetal Bovine Serum, is a serum widely used for culturing various kinds of cells as it contains a lot of growth factors and relatively low concentration of antibodies. It is important that during an experiment to keep the FBS from same stock.

GlutaMAX - is a commercial product that improves cell viability and growth, minimizes ammonia build up to keep the cells healthier.

Pen-Strep - is a mixture of Penicillin and Streptomycin used in cell cultures to as an antibiotic.

The media is already there, we just prepare it by adding supplements to provide an optimized environment for the cells.
This is the end product!








Oh, and make sure to keep the media in the water math before using it. 37.4'C

Intro: 2009 summer internship

Here's some basic info about my 2009 summer internship.

It's at a lab associated with the Manipal Hospital, the Manipal University and Stempeutics, which is referred to as Manipal Institute of Regenerative Medicine (MIRM for short)

Currently, this lab is the only place in India where stemcell research is being done.

The Group that took me in for the summer focuses on Adult Stem Cells, and especially with the Bone marrow mesenchymal stem cells (BMMSC) and Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs), at the moment.

Dr. Jyothi runs this team and Anoop and Shelpa (the senior research staffs) are conducting the research.

Here's Anoop and Shelpa :D

He looks really serious in this picture, but he's actually always happy. He has recently gotten his M. Phill, and has a great sense of humor. He teaches me all sorts of things related to stemcell, but more importantly, he shows me all the different restaurants near MIRM for lunch ;)

Shelpa Also has a degree in Bio-technology, and she teaches me a lot about actual procedures that are done in the lab. She allows me to try them by myself, which is helping me learn a lot faster.

Dr. Jyothi has provided me with essential information about stem cells and in general and about the adult stem cells so I could tag along with Anoop and Shelpa. Since I have only just completed my fisrt year of undergrad, I basically knew nothing. Dr. Jyothi was kind enough to explain most of the concepts herself :D

I couldn't get Dr. Jyothi's picture because she has not yet returned from her buisiness trip out of country. I'll be sure to upload her picture and introduce other staffs in the lab.

and this is me :D, all scrubbed up.