Questions tagged [cell-culture]

Cell culture is the process of growing and maintaining living cells in vitro.

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28
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3answers
19k views

How long does antibiotic-dosed LB maintain good selection?

Various people in our lab will prepare a liter or so of LB, add kanamycin to 25-37 mg/L for selection, and store it at 4 °C for minipreps or other small cultures (where dosing straight LB with a 1000X ...
24
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7answers
6k views

Pipetting damage to cells

I'm curious how much damage is potentially inflicted by shear stress by pipetting. I know that syringes used for stem cell injection can cause a lot of damage. However, to what extent does this happen ...
13
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3answers
48k views

Hoechst or DAPI for nuclear staining?

When is it best to use Hoechst vs. DAPI for nuclear staining? They seem to be very similar on paper. Are there situations where one is clearly preferable?
11
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1answer
5k views

Why are cell lines frozen in vapor phase?

For most of the cell lines I've come across, ATCC recommends storing them in the vapor phase of liquid nitrogen. I'm taking that to mean any place above the level of liquid in the nitrogen storage ...
9
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2answers
2k views

Gloves for Cell Culture?

I always wear gloves when I'm doing cell culture. Moreover, I always spray my gloves with ethanol to disinfect, so that I don't contaminate everything. However, I recently heard the argument that ...
9
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1answer
84 views

What is the effect of exendin on beta-cells?

Do you know if exendin, an analog of GLP-1 (glucagon like peptide-1), can be toxic for beta-cells? For example, what is the effect on INS1 or Min6 cells at a certain concentration or after 90 mins of ...
8
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1answer
3k views

What's the difference between growing cells in culture and cloning them?

The wikipedia page on Hela cells refers to George Gey being able "to isolate one specific cell, multiply it, and start a cell line." Later it says, "In 1955 HeLa cells were the first human cells ...
8
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2answers
2k views

How much time do INS1-E and MIN6 cells need after splitting?

I am currently doing an experiment on cells to test the internalization of a protein. Normally, I seeded my cells the day before the incubation. This worked well for Hela, CHL or PANC1 cells. However, ...
7
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2answers
14k views

How can I resuspend a cell pellet without harming the bacteria?

When using a preculture with Ampicillin in my protein expression, I have to get rid of the preculture medium to avoid carrying over too much beta-lactamases that will destroy the ampicillin in the ...
7
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1answer
654 views

How can I keep HEK cells alive while expressing NMDA receptors?

I am trying to express functional NMDA receptors in HEK293 line cells for single channel recording experiments. The HEK cells are maintained in the standard way (Thomas & Smart 2005) and ...
7
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1answer
4k views

How to reduce/eliminate cell clumping in suspension CHO cells, without using an anti clumping agent?

what is the best way to eliminate clumping suspension cells used for transfection. Anti clumping agents interfere transfection and hence can't be used. Though maintaining the cell density low helps, ...
7
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1answer
259 views

Which human cell lines do not express the GLP-1 receptor?

I need a human cell line that does not express the GLP-1 (glucagon like peptide-1) receptor. I'm working with HeLa cells, do those express the GLP-1 receptor? Which other cell lines exist that don't ...
7
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1answer
489 views

What temperature should mammalian B-Cells be stored at outside of the incubator?

I'm working with murine B-cells. The general protocol is to keep cells on ice to keep them from dying but I've noticed that it makes these cells aggregate and precipitate out. I've heard suggestions ...
7
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1answer
105 views

Providing small molecules to cells on a filter plate

Lets imagine that I have mammalian cells that I've immobilized on a filter. Now I want to keep providing small molecules to these immobilized cells without resolubilizing the cells. The caveat is ...
7
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0answers
885 views

Is there a karyotype database for human cell lines?

I'm looking for the karyotype of a specific cell line. Some karyotypes are well known, such as HeLa or some carcinomas, but some are very hard to find, such as LG2 (B cells). Is there a database ...
6
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2answers
171 views

Why do epithelial cells arrest in response to serum?

Primary epithelial cells, for example human mammary epithelium, fail to proliferate (arrest) in serum-containing medium. Therefore, a common growth medium for epithelium contains pituitary extract ...
6
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1answer
710 views

Measuring algae growth or death

My daughter (10th grade) is doing a science fair project on the toxicity of triclosan for the algae Selenastrum capricornutum. She is wondering the best way to measure the effect, given the limited ...
6
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1answer
3k views

High Glucose vs Low Glucose DMEM for Cell Culture

I've noticed that in mammalian cell culture, there are often two types of DMEM available. High Glucose and Low Glucose. Does it matter which type I use for culturing of cells (e.g. Hela or HEK293)? ...
6
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1answer
13k views

Why is beta-mercaptoethanol often added to cell culture media

Many protocols suggest that beta-mercaptoethanol is necessary for growing cells. It is a reducing agent but what does it mechanistically do for your cells. When would one not add it.
5
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3answers
967 views

Studying effects of alcohol on cells

I am wondering about the logistics of a simple experiment with say 3 types of alcohols, and various concentrations of each, with a control(s) I would like to research the effects of alcohol on ...
5
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3answers
4k views

How do you break up cell clumps when passaging?

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.
5
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2answers
19k views

What does confluency mean?

Since as long as I have been doing cell culture, the word confluency is used to describe the % growth of cells or area covered by them. However, no dictionary that I have found uses this word. I was ...
5
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1answer
1k views

Are HeLa cells edible?

I'm curious if HeLa cells are intrinsically poisonous or dangerous to ingest. My understanding is that some of the contamination in HeLa cells such as HPVs are not readily expressed. I have no plans ...
5
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2answers
3k views

How to convince suspension cells to adhere more tightly?

I'm developing a cell-based assay in 96-well plates that requires adherent cells, as they need to be washed at least twice during the protocol. I'm using in-house strains of HT1080 cells (some ...
5
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1answer
5k views

What's the recommended maximum passage number for mammalian cells (NCI-H441)?

Is there a generally accepted maximum number of times you should split and reseed mammalian culture cells before restarting cultures from new stock?
5
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1answer
325 views

Is cell senescence in culture comparable to that in vivo?

A cell is 'senescent' when is has permanently left the cell-cycle. This can be caused by stresses, or by reaching the 'Hayflick limit' (the cell has reached its replicative lifespan, as defined by its ...
5
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3answers
2k views

Does trypsin strip flask coating?

Mammalian cell/tissue cultures sometimes require flasks coated with proteins. My uneducated guess is that these proteins mimic the ECM, perhaps the basal lamina, so finicky contact-dependent cells can ...
4
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1answer
157 views

Why is glycerol supplied/used as 87% solution?

While preparing glycerol stocks of my transformed bacterial cells (DH5alpha) the protocol I had to follow was that I would make one 50% glycerol stock (500µl culture, 500µl glycerol 50%) and another ...
4
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1answer
1k views

How do I interpret this graph regarding introduced genes and virus-infected cells?

This graph appeared in a practice test for the MCAT. I am trying to interpret it, but it confuses me a bit. On the x-axis we have some introduced genes, and on the y-axis we have % of cells infected. ...
4
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2answers
626 views

What is the smallest oligocelluar organism?

What is the smallest oligocelluar organism? How many cells does it have? EDIT The question is motivated by this comment@Philosophy.SE EDIT as recommended in comments I'm looking for an example of ...
4
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1answer
2k views

Why do some protocols require prewarming a liquid medium before inoculating?

For example, in this protocol for E. coli competent cell preparation, it says: Plate 10 uL E. coli BL21(DE3) cells on a LB-agar plate; incubate overnight (12 hours). Prepare 500 mL SOB medium ...
4
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1answer
17k views

Formula for number of divisions required to form x number of cells (Mitosis and Meiosis)

In case of Mitotic Division, we say that if the total number of cells formed by mitotic divisions are $x$, then counting from the first cell onwards, we get $x-1$ divisions ($N$) $N = x-1$ This is ...
4
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1answer
95 views

Are stem cell lines cryogenically preserved or commercially available like HeLa cells are?

There are HeLa cells, which were taken from sick cancer patient and now is growing worldwide in different laboratories for experiments. HeLa cells can be kept because they are immortal and so they are ...
4
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1answer
232 views

What microscope/magnification would I need to observe P. Acnes bacteria?

I am currently attempting to grow a culture of P. Acnes bacteria. Right now, my only hope in identifying colonies of the bacteria in the culture is to use a black light to find colonies that glow ...
4
votes
1answer
217 views

What does it mean for the signals for transcription and translation to be “conserved”?

I was reading this article: "Overview of vector design for mammalian gene expression." for an explanation of why mammalian cell lines are used for expressing cloned genes, and one of the reasons ...
4
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2answers
2k views

How does formaldehyde/PBS or methanol fixation of cells affect lysosomal pH?

The question is fairly simple - does formaldehyde or methanol fixation in preparation for immunocytochemistry/immunofluorescent staining affect the pH of the lysosomes? Some background: I'm trying to ...
4
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1answer
55 views

Can a cell culture insert plate be used with a vacuum manifold?

There exists 96 well plate setups for use with vacuum manifolds. Is it possible to use 6/12/24 plate inserts with the same setup?
4
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1answer
303 views

How different are tissue-specific fibroblasts from each other?

I am planning to utilize a new system in our lab, in which I will co-culture cancer cells from different tissues with fibroblasts. I have the option to receive skin-derived primary fibroblasts. I ...
4
votes
1answer
106 views

Microinjection intestinal organoids

I am attempting to microinject small intestinal organoids, using the Eppendorf FemtoJet 4i microinjector and Femtotip capillaries, and have come across several issues. This is a new set up in our lab, ...
3
votes
1answer
149 views

What type of flask should I use to culture NTERA2 embryonic cancer stem cells?

I'm just starting my MSc research and I am in the process of making a list of equipments/consumables to order. Is there a specific flask in which I can culture NTERA2 (NTERA2/D1) cell line?
3
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2answers
592 views

Cell line immortalisation biosafety

Cell lines are often immortalised by artificial expression of proteins which specifically cause the knockout of important cancer suppression genes or the activation of proto-oncogenes. Cellular ...
3
votes
1answer
238 views

Looking for protocol to produce a drosophila primary cell line

I'm looking for a general (and easy, this is going to be for an undergrad class) protocol to isolate and culture primary cells from fruit flies.
3
votes
1answer
290 views

How do different tissue culture matrices affect background in fluorescent microscopy?

In response to my previous question, I've been reading up a little bit on poly-D-lysine, Collagen I, Collagen IV, laminin, and other tissue culture coatings that promote cell adhesion. I've always ...
3
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1answer
2k views

Cationic lipid mediated transfection optimization for 150mm dishes

I need to optimize a transfection protocol to transiently express a plasmid encoding a chimera of eyfp attached to the c term of a Golgi apparatus signaling molecule) in hela cells and hepg2 cells ...
3
votes
1answer
1k views

Are cell lines potentially dangerous?

More specifically, if a human subject was exposed to, say, a human cancerous cell line (via intravenous injection or through an open wound, for example), is it possible that they would develop any ...
3
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1answer
119 views

Hela live cell confocal laser scanning - reccommendations for good fluorophore that will show good movement

I've been doing a lot of live cell imaging lately mostly using hela cells expressing some EYFP based chimeric proteins. I'm building a video library for an art student here at the university who is ...
3
votes
1answer
177 views

Is it possible to make anaerobic media too reducing?

I've been struggling to grow an anaerobe (P. gingivalis), I take every precaution to keep the culture conditions free of oxygen, I was wondering if the conditions can be so reducing that they inhibit ...
3
votes
2answers
342 views

What culture medium should we use for tap/drinking water bacteria?

We are a group of high school students from Manila who is performing an investigatory project on a topic that we chose which, at first glance, seemed simple, but turned out a bit complicated. We're ...
3
votes
1answer
83 views

Estimating RPM to RCF in Methods from Older Papers

I'm attempting to replicate a cell biology method from a 1958 Laboratory Investigation paper. The protocol is for the isolation of an extracellular matrix protein, and a key step is a centrifugation ...
3
votes
1answer
527 views

What is the best way to clean plastic flasks that have been used for cell cultures - is virkon a good idea?

When you use cultures e.g. insect cells, which are infected with virus one way to clean the (plastics) shake flasks is with virkon. Which is the most effective way to clean your flasks in order to ...