rhombus

Dear Science noob,

This is an extremely common problem that I have seen over the years. You comment:-

"methods provided in published papers"    .............. this is a big problem in that this part of a paper should probably be the largest section but is commonly just cut to a bear minimum. There are lots of "tricks" that are left out of this section that are essential for thew experiment to be reproduced.

"Same concentration, same cell lines, same conditions"................every cell paper I read states " the cells were grown in a humidified CO2 Incubator at 5% CO2. How many researchers routinely check for humidifiaction AND FYRITE THEIR INCUBATORS TO CHECK PRECISELY THE INTERNAL CO2 CONCENTRATION IN THEIR INCUBATORS.

ARE THE INCUBATORS DIRECT HEAT OR FAN ASSISTED?

WHERE DOE YOUR FOETAL CALF SERUM COME FROM?.......there are massive differences between the qualitof serum sourced for the many countries of the world that produce it.

HOW MANY RESEARCHERS HAVE AUTHENTICATED THEIR CELLS BEFORE USING THEM?

HOW MANY RESEARCHERS ARE WORKING WITH MYCOPLASMA INFECTED CELLS BECAUSE THEY DO NOT TEST THEM?


Does this start to answer your question about reproducibility?

Kindest regards

Uncle Rhombus

Dear metaltemujin,

To answer your question " how to improve your bio skills"

There are many to learn. The most important to start with is basic lab techniques. These may include:-

Basic pipetting skills
Serial Dilutions
Western Blotting Basic cell and tissue culture
Methods of sterilisation
Basic microbiology
Basic molecular biology

.......the list is endless


Then you have to learn about setting up experiments:-

Positive and negative controls
Optimisation steps
The critical path for your experiments
Time management skills
Hypothesis lead experiments
Accuracy as well as precision....i.e. your experiments must be reproducible

......the list is endless

Then you have to learn to present your research/results to your collegues....and then further afield:-

Statistical analysis
Presentation skills
What content to put in AND LEAVE OUT
Anticipating ackward questions
Looking for weaknesses in the results
Further experiments that need to be done

....the list is endless

Then if you are lucky you can contribute or write papers to go into high impsct journals:-

Again most of the above will come into that.

The most important thing I can impress on young people coming into science is the importance of which research group they join. I was very lucky in my career that the first lab head I worked for won a Nobel prize for physiology in the 1980's. This set the standard for the whole department and the post doc I worked for spent many yeras with me teaching me the basics as listed above. You can read many books and scientific papers but the most important thing is

EXPERIENCE....EXPERIENCE.....EXPERIENCE

I hope some of this is useful

Kindest regards

Uncle Rhombus

Dear All,

Uncle Rhombus would like any information from current users of the cell culture system sold by GE Healthcare called WAVE technology. It seems to be a simple system in aiding the bulk up of both insect and mammalian cells in order to produce proteins of interest for high through put screening (HTS)

i.e. Ease of use, technical service back-up, applications, problems in stabilising consistant condition etc etc

It would be much appreciated.

KIndest regards

Uncle Rhombus

spurnima, on 17 July 2012 - 07:51 AM, said:

Dear Spurnima,

The reason(s) for not adding antibiotics are:-

They are very dirty compounds that will affect the cells you are growing

They cover up CRYPTIC CONTAMINATIONS i.e. background levels of contamination that will again affect your cells

If you are using your cells for experimental purposes ......the antibiotics MAY interact in some way with the compounds that you are adding

It is an expensive addition THAT YOU DO NOT NEED TO ADD IN THE FIRST PLACE


Hope this explains some of the good reason not to add antibiotics to your cells in culture

KIndest regards

Uncle Rhombus

jangajarn, on 21 May 2009 - 05:09 PM, said:

Dear Rangajarn,


The most important thing is LISTEN!!!!!!!!!!!!!!!!!!!!!!!!

Then you can start learning

It does help to have people around you who are worth listening to. Be flexible in what you can do practically. There is a process that occurs when doing experiments in science:-
Read about the area you are going to be experimenting in

Try and meet people who are doing/have done similar work....this reduces the time that can sometimes be lost optimising conditions.....its called the "tricks of the trade" which are not to be found in the "Materials and Methods" sections of published papers.

Prepare thoroughly...have I got all the chemicals, consumables, equipment, SOP's that I require.

Do I have the correct supervision by senior staff when I first start. Do I know how to use all the equipment I need to do the experiment. IS THE EQUIPMENT CALIBRATED SO I CAN DO ACCURATE AND PRECISE AND REPRODUCIBLE EXPERIMENTS.

CONCENTRATE ALL YOUR EFFORTS ON 1 THING....... do not be persuaded to "multitask" i.e. give time and effort to get reproducible and accurate experiments that can be repeated, not only by you but by others!!!!!  Once experience THEN multitask....but beware even experienced researchers make errors if put under too much pressure from the PI/Professors.

I personally found the process daunting when I first worked in a research lab in 1983. However over time you will go through the process many times, and like anything in life the more you do it, the easier it becomes.

Hope this gives you some ideas.

Kindest regards

Uncle Rhombus

Biotecmex, on 15 December 2011 - 02:14 PM, said:

The black spot.....is this the spherical black spot attached to the cell???

If it is then this is what I have always described as a "ghost cell"  i.e. all the contents of the cell have been lost. Normally these cells are formed when they do not survive the freezing process. They normally consist of a small percentage of the total.

The only thing I can think of is that all your cells have suffered through the freezing period and that they all circum at different times. This would explain why all your cells have died.

I have these ghost cells in all my newly initiated cells but they disappear over a couple of passages and the viable cells grow exponentially.


If you can get other cells from another batch of frozen stock OR from another lab....then this would be my advice.

Hope this is useful

Kindest regards

Uncle Rhombus

The only way to improve your technique is the old tried and tested way......

Sit down for an hour and practice your pipetting ONTO A PRECISION BALANCE.

You will instantly see with your own eyes IF you have pipetted the correct amount.

It is very important to do this as you can improve:

PRECISION AND ACCURACY.

This is an imperative skill to master as experimentation is dependent upon both of these skills.

Hope this is useful......there are no short cuts to excellence.

Kindest regards

Uncle Rhombus (34 years in the Lab)

ouhscr2, on 05 November 2010 - 07:51 AM, said:

In my opinion it is the only way to grow these cells. We grow both RAW264.7 and J774 (both murine macrophage cell lines) using Techne biological stirrers. My tips are:-

Change the bottle once a month.
Buy Falcon T75 and T175 flasks and use the filtered top on ONE of the side arms of the stirrer. This means that you get adequate gaseous exchange while keeping a barrier to contamination within the incubator.
The stirring speed is very important, 20-25RPM. This will ensure that the cells do not aggregate and settle to the bottom. Too fast and the viability will go down because the cells will be smashing into one another.
Keep the cells at a density between 100,000 and 700,000 cells/ml.
The cells grow extremely fast under these conditions...we try to reduce the growth by using 5-7.5% Foetal Calf Serum.
The Stirrer bottles over time will need to be RESILICONISED. The glass is borosilicate and is used beacause when new the cells do not attach to the glass. However over time, with constant washing and autoclaving, the glass needs to be recoated. This is easy using a siliconising fluid such as REPELCOTE.
Make sure that the silicone seals in the caps are still there and in good condition. This also goes for the rubber that holds the glass stirrer in place.
When preparing an experiment in multiwell plates..it is always best to plate them the day before i.e. give them 24 hours to adhere.

These cells are extremely sensitive to ENDOTOXIN. So be careful with media, FCS/FBS and glassware.

Hope this is useful.

Kindest regards.

Uncle Rhombus.

jakatta70, on Mar 19 2010, 08:42 AM, said:

Dear All,

I apologise to other posters who have read my posts before on Mycoplasma detection and testing, but as this post suggests, there are still some unaware reseachers who are compromising their experiments.

PCR is not a valid test for Mycoplasma. Jakatta70 is completely wrong in what he has stated. The problem with PCR is that it gives too many FALSE NEGATIVES. That means people have tested their cells using PCR and think that they are clean, when really they are using contaminated cells. The Food and Drug Administration (FDA) has not licensed PCR as an approved test for Mycoplasma detection for this reason. It also is a very INSENTIVE test i.e. the cells have to be massively contaminated to get a positive.

"Other methods are time consuming and expensive".....this is complete rubbish. We in our Institute use an independent contracting company  that charge £80/cell line. They use solid agar method as their detection method. This price includes the courier to pick up the cells from us and deliver it to the company. If the cells are contaminated then we are informed of this within 2 DAYS. The test itself however runs over a 4 week period. So to get a definitive negative takes 4 weeks. The reason why it only takes 2 days to get a positive is that it is a very sensitive test so. This means it will pick up a very low contamination. THIS TEST IS APPROVED BY THE FDA.....so if you are taking a drug or being given a vaccination.... THIS IS THE TEST THEY USE.

Finally, the American Tissue Culture Collection (ATCC)...the largest supplier of commercially available cell lines IN THE WORLD.....so they should know what they are talking about.....NO LONGER OFFER PCR AS A MYCOPLASMA DETECTION METHOD.....because of the large number of false negatives you get with this method. They advise using solid agar in combination with Hoescht staining, again the only other FDA approved method. Hoescht again is less sensitive than solid agar and therefore this method will only pick up a medium to high level of mycoplasma contamination.

I hope this explains current thinking on Mycoplasma testing/detection methods. IT IS ONE OF THE MOST IMPORTANT THINGS TO TEST FOR BEFORE DOING ANY SCIENTIFIC RESEARCH USING CELLS. Gene arrays for example can cost many thousands of pound/Euros/Dollars. £80 to test whether a cell is contaminated is peanuts compared to this. You cannot publish data with contaminated cells in any journal that I know of.

I hope you find this advice useful.......32 years of cell culture experience.......and many co author publications.

Kindest regards.

Uncle Rhombus

shyarasu, on Jul 27 2009, 05:58 AM, said:

The detection limit for that assay is 3uM...anything less than that is unreliable. RAW cells can produce huge quantities of NO2-, upto 30-40uM in a 24 hour experiment.

The LPS used is very important and the amount of NO2- produced is dependent upon the source of LPS. In our hands LPS sourced from WS Typhosa gives greater levels of NO2- than LPS derived from E.coli.

The number of cells in your well is also important. If yuo have too few cells then again it will be difficult to measure NO2-.

Your 0uM concentration on your standard curve should not give you much of an OD reading at all. Check what media you are using...this is also important as DMEM has no Nitrate/Nitrite...whereas RPMI has mM amounts of Nitrate. Always subtract the 0uM value from all the other OD...because this is your true baseline reading

Hope this is useful

Kindest regards

Rhombus

biofuel, on Jun 13 2009, 04:46 PM, said:

Having spent 18 years in industry and 13 years in academia...these in my opinion are the pro's and con's

Industry: Pro's
Permanent position.
Training infra-structure
Money and resources
Team atmosphere
Higher Salaries
Better pensions/health cover/SAYE share schemes

Cons:
No academic freedom
Very specialised area of screening/research
Difficulty in getting a position in the first place
Possibility of takeover/merger/relocation........redundancy.


Academia: Pro's
Academic freedom
A more relaxed environment

Con's
Tenured positions/short term contracts
Poor pay structure
Very little career structure
Competition for grant money
Pressure to publish and in good journals
Normally not working in a multi disciplinary environment.

You make your own judgements...but in my opinion go for the industry jobs first.

Kindest regards

Rhombus