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How do you break up cell clumps when passaging?

How do you break up cell clumps when passaging?



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I am working with HepG2 cells, and they really like to form clumps. Pipetting up and down in TrypLE does not seem to be very effective in breaking them up.


I find that I only get clumps (with HeLa and similar cell types) if I leave them in Tryple too long. Generally I leave them in room-temp (not hot) Tryple for ~8 minutes, then I angle the dish, lid off, so that I can see the "sheen" of cells on the dish (this happens in the hood, of course). Then I blast the sheen with Tryple using a 1000ul pipetter, which de-adheres the cells. These are generally much less clumped than if I let the Tryple de-adhere the cells.


In some protocols for setting up primary cultures (for example from mouse bone marrow or rat endometrium), there is a step that requires pushing cell suspension through a large needle to get rid of clumps. Of course, this carries a high risk of damaging the cells, so sometimes collagenase is used instead.

It might also be helpful to wash your cells with PBS without ions before passaging and use tripsin with EDTA. That should get rid of at least some calcium from the plate, so that action of adhesion proteins would be inhibited.


Cell clumps that not dissociate with rigorous pipetting might very well be caused from free DNA in your cell suspension (i.e. if you trypsinized for too long). Free DNA attracts cells which bind altogether forming clumps.

Two possible ways to get rid of these clumps:

  1. Centrifuge your cell suspension at 5000 rpm for 2 minutes with acceleration on (at 4) and brake (at 3) allowing the way heavier molecules to precipitate. Then aspirate ~0.5ml from the top of the suspension and transfer to a new flask. Repeat if needed.
  2. Use DNAse at concentrations from 20-100µg/ml (concentration resulted from personal use) as long as you will not intend to carry out DNA related experiments.

You can always go back and buy a fresh ampule of the cell line you want clumps free :)


Cells: cell structure

b)
-lysozymes,
Hydrolytic enzymes (enzymes that hydrolyse stuff) that that digest material in the cell and recycle it.

-endocytosis,
Lysosomes ingest the broken down crap through endocytosis then use lysozyme to finish the job

b)
-80s,
Bigger than prokaryotes (70s)

-are ribonucleoproteins,
Because they're made of rRNA and proteins

b)
- no ribosomes on surface
That's why it's "smooth"

-maintains the cells turgidity by pushing the chloroplasts to the walls

-capsid/ protein layer beneath envelope

Prokaryotic:
- circular and short strands
- not associated with histones so no chromosomes

2: you can then filter the large pieces of tissue from the homogenate formed

3: put the homogenate in a centrifuge and spin it. The heavy organelle (eg. Nuclei) will form a pellet at the bottom

2: calibrate it to the objective lens (do this by comparing it to a scale on the stage)

The λ of e ̅ is shorter than light so the resolution is much higher

- it must be very very thinly cut costing energy, time, money and potentially creating artefacts


A passage too far?

One study showed that high- and low-passage adenocarcinoma cells had different responses to androgens and retinoids, indicating alterations in gene expression. Researchers in Belgium compared how two strains of LNCaP prostate cancer cells responded to androgens and retinoids, depending on passage numbers. The cells with high passage numbers showed higher-amplitude response curves to 3H-thymidine (measuring cell proliferation), while cells with low passage numbers showed greater growth inhibition by the synthetic androgen R1881, greater PSA mRNA expression and PAP expression (prostatic acid phosphatase). For responses to retinoic acid (atRA), lower-passage cells showed a marked stimulation of 3H-thymidine incorporation in the cells, while lower passage cells only showed growth inhibition. Clearly, passage number affected cellular physiology, which in this case caused the researchers to caution about the use of prostate drugs containing these molecules!


Part 2: The MCAT biology content you need to know for the exam

Note: Clicking on any of these thumbnails will take you to a comprehensive guide on that topic.


Perform the DNA Extraction

  1. Blend together 100 ml of DNA source, 1 ml of salt, and 200 ml of cold water. This takes about 15 seconds on high setting. You are aiming for a homogeneous soupy mixture. The blender breaks apart the cells, releasing the DNA that is stored inside.
  2. Pour the liquid through a strainer into another container. Your goal is to remove the large solid particles. Keep the liquid discard the solids.
  3. Add 30 ml liquid detergent to the liquid. Stir or swirl the liquid to mix it. Allow this solution to react for 5-10 minutes before proceeding to the next step.
  4. Add a small pinch of meat tenderizer or a squirt of pineapple juice or contact lens cleaner solution to each vial or tube. Swirl the contents gently to incorporate the enzyme. Harsh stirring will break the DNA and make it harder to see in the container.
  5. Tilt each tube and pour alcohol down the side of each glass or plastic to form a floating layer on top of the liquid. Alcohol is less dense than water, so it will float on the liquid, but you don't want to pour it into the tubes because then it will mix. If you examine the interface between the alcohol and each sample, you should see a white stringy mass. This is the DNA!
  6. Use a wooden skewer or a straw to capture and collect the DNA from each tube. You can examine the DNA using a microscope or magnifying glass or place it in a small container of alcohol to save it.

5-day blastocyst implantation timeline

Successful implantation of the blastocyst in the uterus is necessary for the fetus to grow. For many women afflicted with infertility, this step is compromised. Implantation rates also decline with female age due to a rising chance of chromosomal abnormality. Successful in vitro fertilization (IVF) transfer rates are about 37.1% for women under the age of 35, and lessen with age. Normally, a human embryo will take four days to travel down the fallopian tube and into the uterine lining. During IVF, implantation will occur between six to ten days after egg retrieval. This is also one to five days after a blastocyst transfer in the recipient.

A summary timeline of normal implantation follows:

  1. Day 0: the egg is fertilized high up in the fallopian tubes and forms the zona pellucida
  2. Day 1-3: the egg undergoes division in the solid morula stage
  3. Day 1-4: the egg continues to move down the fallopian tube and into the uterus
  4. Day 5: the egg has begun to transform into a hollow blastocyst, and has traveled and implanted itself into the uterine wall near a source of blood (future food!)
  5. Day 5-9: the blastocyst continues to divide, and still has the outer trophoblast layer, the fluid cavity, and inner cell mass that are the stem cells that will form future fetal tissue

How do you break up cell clumps when passaging? - Biology

Cells from the lining of your mouth come loose easily, so you will be able to collect cells containing your DNA by swishing a liquid around in your mouth.

The cells from the lining of your mouth also come off whenever you chew food. How do you think your body replaces the cells that come off the lining of your mouth when you eat?

To extract DNA from your cells, you will need to separate the DNA from the other types of biological molecules in your cells. What are the other main types of large biological molecules in cells?

You will be using the same basic steps that biologists use when they extract DNA (e.g. to clone DNA or to make a DNA fingerprint). You will follow these 3 easy steps to extract the DNA:

D etergent
E N zymes (meat tenderizer)
A lcohol

Getting Your Sample of Cells

Obtain a cup with sports drink. You will need to get thousands of your cheek cells in the sports drink in order to extract enough DNA to see. Therefore you should swish the sports drink around in your mouth vigorously for at least one minute. Then spit the drink back into the cup.

Add a small amount of detergent to a test tube (about 0.25 mL or 1 drops). Now carefully pour the drink containing your cheek cells into the test tube with detergent until the tube is half full .

Why am I adding detergent?

To get the DNA out of your cheek cells you need to break open both the cell membranes and the nuclear membranes. Cell membranes and nuclear membranes consist primarily of lipids. Dishwashing detergent, like all soaps, breaks up lipids. This is why you use detergents to remove fats (which are lipids) from dirty dishes. Adding the detergent to you cheek cell solution will break open the cell membranes and nuclear membranes and release your DNA into the solution.

Add a pinch of enzyme (meat tenderizer) to your test tube. To mix the tube gently tap the bottom of the test tube 20 times. Let the mixture sit for at least 10 minutes. While you are waiting, you will learn about the structure of DNA. Read through the hand out titled “DNA Structure”.

Using a pipette, slowly add cold rubbing alcohol into the test tube let the alcohol run down the side of the test tube so it forms a layer on top of the soapy liquid. Add alcohol until you have about 2 cm of alcohol in the tube. Alcohol is less dense than water, so it floats on top. Do not mix or bump the test tube for 10 minutes. DNA molecules will clump together where the soapy water below meets the cold alcohol above, and you will be able to see these clumps of DNA as white strands. While you are waiting for the DNA to become visible you will learn about DNA replication.

Why am I adding alcohol? The cold alcohol reduces the solubility of DNA. When cold alcohol is poured on top of the solution, the DNA precipitates out into the alcohol layer, while the lipids and proteins stay in the solution.

As you can see in the figure below, DNA consists of two strands of nucleotides wound together in a spiral called a double helix . Each nucleotide contains a phosphate and a sugar molecule called a deoxyribose (which explains why the complete name for DNA is deoxyribonucleic acid). Each nucleotide also has one of four different nitrogenous bases: adenine ( A ), thymine ( T ), guanine ( G ), and cytosine ( C ).

(Adapted from Figure 9.4 in Biology by Johnson and Raven)

The drawings below show a very small section of the DNA double helix from three very different organisms: a plant, a mammal, and a bacterium. Each strand of DNA shown contains five nucleotides, each with a:

S = five carbon sugar molecule called deoxyribose

A = adenine, C = cytosine, G = guanine, or T = thymine, the DNA nucleotide bases

You can see that the phosphate from one nucleotide is bonded to the sugar in the next nucleotide to form the backbone of each strand in the DNA molecule. The bases of the nucleotides in each strand of DNA extend toward each other in the center of the DNA double helix molecule. A crucial aspect of DNA structure is the base-pairing rule : A in one strand always pairs with T in the other strand, and G in one strand always pairs with C in the other strand. You will see later that this base-pairing is crucial for the cell to make new copies of each DNA molecule in preparation for cell division.

Which characteristics are similar in the DNA of plants, mammals and bacteria? What is the only characteristic that differs between these segments of DNA from a plant, a mammal and a bacterium? These observations illustrate the similarity of the basic structure of DNA in all living organisms. The genetic differences between plants, mammals and bacteria are due to differences in the sequence of bases in their DNA.

Cells in our body are dividing all the time. For example, cell division in the lining of your mouth provides the replacements for the cells that come off whenever you chew food. Before a cell can divide, the cell must make a copy of all the DNA in each chromosome this process is called DNA replication . Why is DNA replication necessary before each cell division?

As shown in the figure below, the first step in DNA replication is the separation of the two strands of the DNA double helix by the enzyme DNA helicase . After the two strands are separated, another enzyme, DNA polymerase , forms a new matching DNA strand for each of the old DNA strands. DNA polymerase forms the new matching DNA strand by adding nucleotides one at a time and joining each new nucleotide to the previous nucleotide in the growing DNA strand. Each nucleotide added to the new strand of DNA follows the base-pairing rule with the matching nucleotide on the old strand of DNA. The result is two identical DNA double helixes.

(Adapted from Figure 9.9 in Biology by Johnson and Raven)

In the drawing below, the small segment of plant DNA (from page 3) is shown after the two strands of the DNA molecule have been separated by DNA helicase. Your job is to play the role of DNA polymerase and create the new matching strands of DNA to make two pieces of double-stranded DNA in the drawing below. Use the base-pairing rule to determine which nucleotides to add.

During actual DNA replication sometimes mistakes are made and the wrong nucleotide is added to the new strand of DNA. DNA polymerase can “proofread” each new double helix DNA strand for mistakes and backtrack to fix any mistakes it finds. To fix a mistake it finds, DNA polymerase removes the incorrectly paired nucleotide and replaces it with the correct one. If a mistake is made and not found, the mistake can become permanent. Then, any daughter cells will have this same change in the DNA molecule. These changes are called point mutations because they change the genetic code at one point, i.e. one nucleotide. Point mutations can result in significant effects, such as the genetic disease, sickle cell anemia.

Answer the following questions in well thought out complete sentences. DO NOT START A SENTENCE WITH “IT”, “THEY” or “BECAUSE”.

Why is DNA so important in biology?

What is the function of DNA?

Where is DNA found in our bodies?

Compare the sugar-phosphate arrangement in the backbone of the DNA from the plant, the mammal and the bacterium. Are there any differences?

Which bases are present in the DNA of the plant? The mammal? The bacterium?

Are the same bases present in all three cases?

Are the bases in the same order?

Describe the pattern of base pair matching for the two strands in the plant's DNA. In other words, which types of bases are paired together? Does the DNA from the mammal follow the same base-pairing rule as the DNA from the plant? Is base-pairing the same or different in the DNA of the bacterium?

7. Which of the following do you think will contain DNA? Explain your reasoning .

bananas __ concrete __ fossils __ meat __ metal __ spinach __ strawberries __

8. Describe the function of DNA polymerase. Explain why each part of the name DNA polymerase (DNA, polymer, -ase) makes sense.


Invasion Assay Using 3D Matrices

The extracellular matrix (ECM) is a network of molecules that provide a structural framework for cells and tissues and helps facilitate intercellular communication. Three-dimensional cell culture techniques have been developed to more accurately model this extracellular environment for in vitro study. While many cell processes during migration through 3D matrices are similar to those required for movement across rigid 2D surfaces, including adherence, migration through ECM also requires cells to modulate and invade this polymeric-mesh of ECM.

In this video, we will present the structure and function of ECM and the basic mechanisms of how cells migrate through it. Then, we will examine the protocol of an assay for tube formation by endothelial cells, whose steps can be generalized to other experiments based on 3D matrices. We will finish by exploring several other biological questions that can be addressed using ECM invasion assays.

Procedure

Scientists have developed 3D models to more accurately study cell invasion and migration processes. While most traditional cell culture systems are 2D, cells in our tissues exist within a 3D network of molecules known as the extracellular matrix or ECM. While many of the mechanistic processes required for cell motility in 2D and 3D are similar, factors such as the reduced stiffness of ECM compared to plastic surfaces, the addition of a third dimension for migration, and the physical hindrance of moving through the mesh of long polymers in the ECM, all present different challenges to the cell compared to two-dimensional migration.

This video will briefly introduce the basic function and structure of the ECM, as well as the mechanisms by which cells modulate and migrate through it. Next, we’ll discuss a general protocol used to study endothelial cell invasion. Finally, we will highlight several applications of 3D matrices to studying different biological questions.

Let’s begin by examining the composition of the ECM, and how cells interact with it.

The ECM performs many functions, such as providing support for cells, facilitating intercellular communication, and separating tissues. ECM composition varies among different tissues and has different biological properties, but it can be classified into two broad types. The basement membrane serves to anchor and separate tissues, while interstitial matrix surrounds and supports the cells within a tissue. The interstitial matrix is mostly composed of the fibrous protein collagen, but also includes elastin and fibronectin.

Several biological processes need to occur for cells to migrate through the ECM. The first is cell-matrix adhesion, which involves transmembrane proteins called integrins. These link the ECM to the cell’s internal scaffold, known as the cytoskeleton.

Another process is the structural rearrangement of the cell’s cytoskeleton. This leads to the formation of specialized structures called invadopodia, which are protrusions of the cell into its surrounding matrix. The final step is ECM modulation. This typically involves degradative molecules known as matrix metalloproteases or MMPs, which accumulate in the invadopodia and degrade the surrounding ECM, facilitating cell invasion. 3D matrix invasion assays allow scientists to visualize and study this complex process.

Now that you’re familiar with ECM and its interaction with cells, let’s walk through a protocol for studying ECM invasion by endothelial cells to form tubules. By culturing endothelial cells in a 3D environment, one can simulate the biological process of blood vessel growth, also known as angiogenesis, which is important during both normal development, as well as cancer.

First, endothelial cells are cultured, and a single cell suspension is prepared by treating the cells with proteases such as trypsin, and passing them through a mesh filter to break up cell clumps. The 3D matrix, commonly composed of collagen, fibrin, laminin, or more complex combinations of these components—which can either be prepared in-lab or ordered from commercial vendors—is then thawed on ice. Since most ECM preparations polymerize at higher temperatures, it is helpful to keep other equipment and reagents cold as well. The cell suspension is mixed with the thawed matrix solution to embed cells, and this mixture is placed into a cell culture incubator where the higher temperature will cause the matrix to polymerize.

Once the cell-containing matrix is set, culture media containing angiogenic factors is added to the matrix dish. Using time-lapse microscopy software, individual cells can then be tracked to observe their migration through the matrix. The resulting images are analyzed, and cell positions are used to calculate movement direction and distance in microns. These values can then be plotted to determine locomotory activity—the average migration rate of the cells. Finally, tube network formation is observed and analyzed using visualization software to identify features such as nodes, tubes, and loops.

Now, let’s explore a few applications of 3D matrices in specific experiments.

Cell migration is mediated by active modulation of the cellular cytoskeleton. In this experiment, collagen matrices were prepared and mixed with a stain containing red fluorescent protein to allow for visualization. Individual cell spheroids, which are free-floating cell clusters, were isolated and embedded in the collagen matrix. Following incubation, the embedded cells were stained for specific cytoskeletal components, and imaged by fluorescence microscopy. Researchers observed cytoskeletal components and their alterations as cells migrated through the ECM.

Scientists can also study how the properties of the ECM affect migration. Using a concentric gel system, where cells are embedded in an inner gel matrix surrounded by outer matrices of varying concentrations, scientists can track cells using time-lapse microscopy to study their migration from the inner gel to the initially cell-free outer gel. Researchers observed that the greater stiffness of higher concentration gels resulted in increases in both cell displacement and overall distance of cell migration.

Finally, matrix invasion assays can be performed within a living animal to study angiogenesis in an organ-specific context. Here, fibrin gels—commonly used in tissue engineering due to their biodegradable nature—were generated, followed by implantation into mouse lungs where the gels were held in place by a “glue” made of the protein fibrinogen. Cell migration and new blood vessel formation were allowed to occur for the following 7 to 30 days, after which the lungs and fibrin gels were harvested, fixed, and sectioned. Imaging of these sections revealed blood vessel and alveoli formation in the implanted gels, giving researchers insight into this crucial aspect of lung development in its in vivo setting.

You’ve just watched JoVE’s video on extracellular matrix invasion assays. This video discussed the composition of the ECM and how cells migrate through it, presented a simple protocol to study endothelial cell migration through a 3D matrix, and highlighted several cellular processes currently being studied in the context of cell-ECM interactions. Because endogenous cell migration occurs in 3D space, these biological conditions are best simulated by 3D culture techniques. Improvements in matrix composition will continue to allow scientists to more accurately replicate and study cellular migration in the lab. As always, thanks for watching!


Ion Transport Mechanisms

There are several ion transport mechanisms within the cell membrane that function to maintain proper levels of solutes inside and outside the cell. One of the most important is the sodium-potassium ATPase pump. This system uses the energy stored in ATP to pump potassium into the cell and sodium out of the cell. Another critical pump is the calcium ATPase pump which moves calcium out of the cell or pumps it into the endoplasmic reticulum. This transfer of ions back and forth across the membrane creates a membrane potential that drives the ionic currents. Also, water moves in and out of the cell based on the differences in the ion concentrations. This way, ion transport helps to regulate both the volume of the cell and the membrane potential.


The image above shows the components of a sodium-potassium pump in the phospholipid bilayer of the cell membrane.


Procedure

    Obtain a uniform suspension of cells: Follow the typsinization/trypsin neutralization protocol for the specific cell type. Place the cell suspension in a suitably-sized conical centrifuge tube. For an accurate cell count to be obtained, a uniform suspension containing single cells is necessary. Pipette the cell suspension up and down in the tube 5-7 times using a pipette with a small bore (5 ml or 10 ml pipette). For cells thawed from cryopreservation (in 1ml cryopreservation medium), pipette up and down 7-10 times using a one ml pipette.

For an accurate determination, the total number of cells overlying one 1 mm 2 should be between 15 and 50. If the number of cells per 1 mm 2 exceeds 50, dilute the sample and count again. If the number of cells per 1 mm 2 is less than 15, use a less diluted sample. If less dilute samples are not available, count cells on both sides of the hemocytometer (8 x 1 mm 2 areas).

Keep a separate count of viable and non-viable cells. If more than 25% of cells are non-viable, the culture is not being maintained on the appropriate amount of media. Reincubate the culture and adjust the volume of media according to the confluency of the cells and the appearance of the media. Include cells on top and left touching middle line. The cells touching middle line at bottom and right are not counted.

i. Trypan Blue is the "vital stain" excluded from live cells.
ii. Live cells appear colourless and bright (refractile) under phase contrast.
iii. Dead cells stain blue and are non-refractile.

  • %Cell Viability = [Total Viable cells (Unstained) / Total cells (Viable +Dead)] X 100.
  • Viable Cells/ml = Average viable cell count per square x Dilution Factor x 10 4 /
  • Average viable cell count per square = Total number of viable cells in 4 squares / 4.
  • Dilution Factor = Total Volume (Volume of sample + Volume of diluting liquid) / Volume of sample.
  • Total viable cells/Sample = Viable Cells/ml x The original volume of fluid from which the cell sample was removed.
  • Volume of media needed = (Number of cells needed/Total number of viable cells) x 1000.


Watch the video: Πολυώνυμα: Διαίρεση Αλγοριθμική (August 2022).