Cell Fractionation

Introduction:
This experiment was aimed to learn how to do cell fraction or Sucrose centrifugation, observe cell organelles by using specific dyes and calculate DNA concentration.
Differential centrifugation is common method to separate organelles of cell from cytosol and membrane. The separation of a specific organelle is based on differences of segmentation rate of organelles. This rate is related with size, shape and density of the organelle. 1,2 and 3
Sucrose gradient centrifugation is used to achieve purer cellular fractionation mostly. Sucrose gradient forms layers to separate fractions of each other and it helps clearer difference between layers. Sucrose gradient centrifugation is used often for cellular fractionation.1,2 and 3
Nucleus is a cellular organelle being enclosed and having genetic material of cell in eukaryotic cells. Nucleus is a control part of cell and does this mission by gene expression. This gene expression regulates cell organization. 3
Mitochondrion is a kind of energy convertor for all living eukaryotic cells. Mitochondria are thought of they evolved from bacteria because of their own membrane, DNA, RNA, ribosomes and this antique symbiotic relationship formed an organelle for first eukaryotic cells. Eukaryotic cells generate many of ATP by using reactions in mitochondria. These reactions are formed between membranes of mitochondria. Enzymes and transporter on membranes served this production.3, 4
Spectrophotometry is a measurement technique for quantitate by reflection of specific wavelength of a material. Each material has specific electron order. Spectrophotometry sends a beam having a certain wavelength and this beam induce electron to higher level by giving energy.5,6
Two dyes were used for this experiment, aceto-orcien and Janus green B. Aceto-orcien is dye for staining chromatins and we can see nucleus by this method. Janus Green B stain mitochondria and it can show mitochondria activity. When oxygen is present, dyed mitochondria looks pink; when oxygen is absent, mitochondria looks blue.5,6
Material and methods:
Part 1:
Firstly, the liver piece was cut to small portions. Some of the small portions were placed into mortal to smash by petrel with 10 ml of 0.25M sucrose solution until solids were disappeared. The sample was transformed into a falcon tubes to perform in centrifuge for 5 minutes at 800 rpm. 5 ml of the supernatant was separated from tube and kept in Tube H (Homogenate). The residual of homogenate was centrifuged again at 5000 rpm for 15 min. After centrifuging, we had a nuclear pellet and then 5 ml of 0.25M sucrose solution was added into the tube for suspending. The nuclear fraction was separated in Tube N by labelling. Centrifuge process was used again at 10000 rpm for 30 min to achieve. After resuspending mitochondrial pellet with 0.25M sucrose, we kept the solution and we labelled as Tube M for mitochondrial fraction. All samples (Tube H, N, and M) were placed into fridge (at about 200 C). This cold condition helps for stabilizing liver enzymes by inactiving.
Part 2:
1 ml 0,25 M sucrose was used for filling to 15 eppendorf tubes. 5 tubes were labelled as H1, H2, H3, H4 and H5 for homogenate fractions. N1, N2, N3, N4 and N5 terms as labels are used for nuclear fraction. Same process was done for mitochondrial fractions. Initial eppendorf tubes were used as stock solution containing 1.25 ml and for tubes H, N and M. 1.25 ml of Tube H was put into Tube H2 and then mixed by using micropipette gently. Same process was used from 1.25 ml of Tube H2 to Tube H3. This processes repeated for all Tube Hs and Ns and Ms. It is called as serial dilution. 250 µl samples were taken from each tube to place into plates for spectrophotometer analysis. Next step, samples were performed under light microscopes by using dyes. Small amount of Tube N was taken and Aceto orcein was added onto slides and then cover slide was closed gently against bubbles for observation of nuclear fraction. Tube M was performed under microscope by using Janus Green B.
Result:
⦁ The value of A260= [Each value from spectrophotometer]-[Average of blank] :
Average of blank= (3,575+3,573+3,380+3,463+3,475+3,458)/6=3,487
Tubes H 0.25M sucrose Previous tube Final volume Final dilution factor [Each value from spectrophotometer]-[Average of blank] 260nm absorption:
1 1.25 ml Stock Solution No result No result
2 1 ml 0.25 ml 1.25 ml 1/5 No result No result
3 1 ml 0.25 ml 1.25 ml 1/25 3,637-3,487 0,211
4 1 ml 0.25 ml 1.25 ml 1/125 3,549-3,487 0,15
5 1 ml 0.25 ml 1.25 ml 1/625 3,549-3,487 0,062
6 1 ml 0.25 ml 1.25 ml 1/3125 3,478-3,487 -0,009
Table 1.1- Values of absorption for tubes H
Tubes N 0.25M sucrose Previous tube Final volume Final dilution factor [Each value from spectrophotometer]-[Average of blank] 260nm absorption:
1 1.25 ml Stock Solution 3,466-3,487 -0,021
2 1 ml 0.25 ml 1.25 ml 1/5 3,403-3,487 -0,084
3 1 ml 0.25 ml 1.25 ml 1/25 3,501-3,487 0,014
4 1 ml 0.25 ml 1.25 ml 1/125 3,529-3,487 0,042
5 1 ml 0.25 ml 1.25 ml 1/625 3,543-3,487 0,056
6 1 ml 0.25 ml 1.25 ml 1/3125 3,629-3,487 0,142
Table 1.2- Values of absorption for tubes N
Tubes M 0.25M sucrose Previous tube Final volume Final dilution factor [Each value from spectrophotometer]-[Average of blank] 260nm absorption:
1 1.25 ml Stock Solution 3,357-3,487 -0,13
2 1 ml 0.25 ml 1.25 ml 1/5 3,476-3,487 -0,011
3 1 ml 0.25 ml 1.25 ml 1/25 3,609-3,487 0,122
4 1 ml 0.25 ml 1.25 ml 1/125 3,571-3,487 0,084
5 1 ml 0.25 ml 1.25 ml 1/625 3,582-3,487 0,095
6 1 ml 0.25 ml 1.25 ml 1/3125 3,573-3,487 0,086
Table 1.3-Values of absorption for tubes M
⦁ DNA concentration (µg/ml) =A260*50µg/ml*dilution factor
Tubes H DNA concentration
1 No result
2 No result
3 389,7727
4 753,2076
5 846,2713
6 -333,93
Table 2.1-DNA concentrations of homogenate fractions
Tubes N DNA concentration
1 -5,25
2 -57,0839
3 25,8617
4 210,8981
5 764,3741
6 5268,667
Tubes M DNA concentration
1 -32,5
2 -7,47528
3 225,3662
4 421,7963
5 1296,706
6 3190,883
Discussion:
Part1:
Cellular fraction was done according to procedure. In this part, as a difference, we separate last solution and kept it too. Because mitochondria can cause un separated complex and it can cause a wrong observation under microscope. We kept some M fraction for next part of experiment for any issue.
Part 2:
Fractions were performed according to how they have DNA by using spectrophotometer with 260 nm wavelengths. Then we calculated using DNA concentration formula. However we needed 260 nm absorption values. We used blank samples to calculate absorption better. We took average and subtracted from the value readied to find real absorption for DNA. When some values were investigated, these can be unexpected. Because these values can’t be expected negative. We can think of samples have been contaminated. Some contaminant could cause a change of absorption values. This is best explanation for negative results. Another reason can be device. The device has readied measurements. (Table 1.1, 1.2 and 1.3)
According to the results of the DNA concentration after calculations, the concentration increases without negative values coming from first tables. If we investigate right values, serial dilution can provide denser DNA concentration.
Finally dyes were used for last observation. Aceto-orcien couldn’t give a clear image, because of pellets and some contaminants prevented (Figure 3.1 and 3.2). We couldn’t achieve taking good observations under 10x and 40x. At least Janus Green B gave a clear image for mitochondria (Figure 3.3). Blue colour could be seen easily. This blue colour says to us that mitochondria are dead. At oxygen absent, Janus green B gives blue colour.
References:
[1]Chapter 3: Cell Fractionation – Introduction, Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College
[2]De Duve, C. Exploring cells with a centrifuge. Nobel Lecture. 1974
[3]Lodish, H; Berk A; Matsudaira P; Kaiser CA; Krieger M; Scott MP; Zipursky SL; Darnell J. (2004). Molecular Cell Biology (5th ed.). New York: WH Freeman.
[4]Henze K, Martin W; Martin, William (2003). “Evolutionary biology: essence of mitochondria”. Nature. 426: 127-8.
[5]Allen, D., Cooksey, C., & Tsai, B. (2010, October 5). Spectrophotometry. Retrieved (from http://www.nist.gov/pml/div685/grp03/spectrophotometry.cfm)
[6]McBride HM, Neuspiel M, Wasiak S (2006). “Mitochondria: more than just a powerhouse”. Curr. Biol. 16 . (doi:10.1016/j.cub.2006.06.054).

Cytoskeleton Components

Introduction:
For this experiment, the object is to observe cytoskeleton components of breast cells (MCF7) under microscope by using the method immunostaining.
Cytoskeleton:
Cytoskeleton is structure that give stabile shape and movement for cells. This cytoskeleton structure is made out of polymers of various subunits. Major kinds of cytoskeleton are microfilaments, microtubule and intermediate filaments. (1)
Microfilaments as also known actin microfilaments are helical polymers being made of actin proteins. They have flexibility and also they are located beneath the plasma membrane intensively. One of the most important duties of these filaments are movement of cell. Such as muscles cells utilize these filaments. (2)
Microtubules are long and hollow tubules and consist of the protein tubulin, called α-tubulin and ß-tubulin. Tubules have a much stiffer structure than actin filaments. Tubulin can be in all eukaryotic cells and can be in multiple isoforms. Organelles and vesicles can move inside cell by using microtubules and some motor protein. The tubules are dispersed in the cytoplasm and spread into the cell like a network. Mitotic spindle and flagella and cilia of bacteria are made of microtubules. (3)

Intermediate filaments are polymers looking like rope-like. They are called intermediate because of their length is between tubules and actin filaments. Intermediate filaments are formed by intermediate proteins and filaments have various forms according to specialized cells.

Cell Culture:

Scientists need cell culture to study on different cells. Cell culture is provided by mimicking the environmental conditions for the cells. If cells are removed from a living organisms, this process are named as in vitro (in dish). Opposite of use of this process is in vivo (in life). Cell culture has already difficulty even when all conditions are suit for cells. Contamination is important factor for cell culture. Other kinds of organisms can place and grow in plate such as fungus, bacteria. Cell can be formed by using various methods:
Primary cells: culture is formed from tissue of a living organism directly.
Secondary cells: Cells that can produce thousands generations with appreciate conditions.
Immortalized cells: Cancer cells that are used for study such as HeLa. They are immortalized, because cancer cells divide unstoppably.
Cell cultures can be used for cell physiology, observing effects of molecules to cells, producing tissue and synthesizing biological molecules in laboratories.
Cell cultures have some negative effects for studies because of artificial conditions they have. Tissue cells can’t adapt to conditions and different enzymatic activity can occur. Reality and cell culture can’t be same results.

Materials and Methods:
Firstly medium was removed from plate by using micropipette. Removing medium by micropipette was done from the wall of plate carefully to not take cells on coverslip. 1 ml of 1x PBS was used for the process washing cells for 2 times and washing with PBS was done slowly from the wall of the plate. 0,5 ml of the PFA in cold water beaker was added for fixing cells, removing nonsuit cells for observation. For 20 min and at RT (room temperature), the sample has been waited for effect of PFA. The sample was washed again with PBS for 3 times to detract PFA from the sample and then 1 ml of 1% TrintonX-100/PBS was added to allow the dye to enter the cells to permeabilize cells by putting from the wall again. The sample has been waited for permeability for 15 min at RT.
During waiting, we started to prepare a block solution. For solution, 1 ml of 1% Trintox-100 was taken and next 0,03 g of BSA was measured on balance for achieving 3% BSA in TrintonX-100 solution.
0,5 ml of this block solution was added into the plate and it has been waited for 30 min at RT to show its effect. After waiting for 30 min, it was removed from plate. Fluorescently conjugated Alexa 488-conjugated Phalloidin in 3% BSA/0,1% TritonX-100 for 30 min in dark were incubated to cells with 200µl of it. By tweezer, cover glass including cells was turned down and placed on the dish separated 4 parts for each group. This dish includes paraffin for cells attach to ground. Cells were washed with PBS-TrintonX for 2 times and 10 min in dark to remove completely unbound antibiotics from the media. After washing, dish was covered with foil. DAPI was dropped onto other slide, after slide was dried by dH2O. The Glass with cells was put onto slide with DAPI. After mounting cells by glue with nail polisher, observation under microscope was processed.
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Discussion:
In this experiment, we observed microfilaments and nuclues of breast cancer cells. We used various dyes for observing and antibiotics(DPI, Fluorescently conjugated Phalloidin).
Firstly, the images (Figure 1.2 and 2.1) can reflect 2 main structures of cells, plasma membrane and nucleus. Small and dense cells can be seen as difference than cells of other group, because these breast cancer cells can divide uncontrolly and this couses small and dense cell colonies.
We used secondry antibodies for a better observations, because primary antibodies have chance to not bind firmly or to bind wrong places. As secondary antibodies bind primary antibodies that already have bound correctly, it serves better results. This forms an image of denser dye under microscope.
Actin filaments or microfilaments are found under plasma membrane in high concenration. Because of that, we can see shape of cells. In images(Figure 2.1 and 2.2), green lines show microfilamets and naturally plasma membrane. Phalloidin was used as a secondary antibody and bound to receptors. Its fuolorescent feature provide image under microscope. We can notice some nested regions for green parts. The reason of that can be that dye couldn’t have distubeted well. At any point of stages, this could occur.
DPI is a dye that is used for staining nuclues. DNA (or nucleus) with DAPI is seen as blue under fluorosence microscope (Figure 1.3 and 2.3). DAPI molecules pass through membrane and bind to pockets of DNA molecules. Fuolorecently DAPI can give bright under fluorosence microscope.
References:
(1) McKinley, Michael; Dean O’Loughlin, Valerie; Pennefather-O’Brien, Elizabeth; Harris, Ronald .2015. Human Anatomy .4th edition. New York: McGraw Hill Education. p. 29.
(2) Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter. Molecular Biology of the Cell 6th Edition. Garland Science. (November 19, 2014). p.898
(3) Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter. Molecular Biology of the Cell 6th Edition. Garland Science. (November 19, 2014). p.926
(4) Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter. Molecular Biology of the Cell 6th Edition. Garland Science. (November 19, 2014). p.944
(5) Arshad Chaudhry. CELL CULTURE. SCQ/THE SCIENCE CREATIVE QUARTERLY. August 2004.