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dw237606's Content

There have been 25 items by dw237606 (Search limited from 22-February 19)

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#116008 Yeast contamination in one well

Posted by dw237606 on 25 July 2011 - 03:21 PM in Tissue and Cell Culture

I would toss the entire plate out to avoid the risk of it contaminating the other wells.

#111806 MTT problems with Huh7 cell line, plzzzzzzz help

Posted by dw237606 on 03 June 2011 - 04:49 PM in MTT, Proliferation and Cytotoxicity Assay

I'm sure you have already thought of this and I don't mean to insult your intelligence but something else that might be worth trying is solubilizing the formazan in 100 uL of DMSO instead of 200 in order to magnify any changes in dye content in each well.

What are the 3 drugs you have tried and at what concentrations?

#111805 MTT problems with Huh7 cell line, plzzzzzzz help

Posted by dw237606 on 03 June 2011 - 04:45 PM in MTT, Proliferation and Cytotoxicity Assay

Have you tried using media that contains serum? In the past I have had problems with certain cell lines when using serum free medium but I have no experience with this cell line.

Are you using 0.025mg/mL of MTT or 0.025 mg in 5 mL (making it 0.005 mg/mL)? I highly doubt this is the source of the problem but I have always used 3-5 mg/mL of MTT, and put 50 uL of the MTT solution along with 100 uL of complete media and allowed it incubate for 4 hours before aspirating and stabilizing in DMSO.

For a positive control the standard way that I have seen done is as follows - this is probably the way you are already doing it but just in case I'll outline the procedure here in detail:

Plate the same number of cells in the wells as you are for the wells that will receive treatments.
After 24 hours aspirate the medium the same as all the other wells, and replace it with with fresh, complete medium.
After incubating for however long, aspirate the medium (same as all the other wells) and replace with the medium/MTT solution describe above.
Incubate for 4 hours, aspirate the medium/MTT solution, solubilize the dye in DMSO (100 uL) and then add 15 uL of 0.1 uM glycine buffer (pH 10.5)
Read the absorbance.

#110008 Help ID the contamination

Posted by dw237606 on 16 May 2011 - 01:01 PM in Tissue and Cell Culture

Am I correct in my assessment that this is a fungus or yeast?

In the past it has proven to be unresponsive to 2.5 ug/mL amphotericin-B.

#110007 Help ID the contamination

Posted by dw237606 on 16 May 2011 - 12:59 PM in Tissue and Cell Culture

1) The system is a central AC unit. The other labs are working with various cancer cell lines, neurons, and blood brain barriar cells none of which have a morphology anything like what is seen here. However, I do know that there was some contamination that occured in another lab's cell lines (that I share an incubator with) a little over 2 weeks ago. I am not sure as to what the contamination was - all I know is that they prepped new media solutions and disposed of the infected cultures and the problem went away. While it is extremely plausible that this is directly related to my contamination I have some doubts due to the fact that there are about a dozen or so other flasks being cultured in the same incubator and none of these have become contaminated. Also, I always do my cell work first thing in the morning - before anyone else making the chance of carry over from the contaminated cells to mine very unlikely (along with the standard asceptic technique).

2) The only thing I know has changed in the past 2 weeks in reference to cell culture is I began using a new pack of cell culture flasks after the last set ran out. These new flasks were purchased as sterile, and I autoclaved them just to be sure and yet this still occured making me doubt that this is related to the contamination.

3) All equipment is sterilized by 70% ethanol prior to use and I do this religiously to every piece of equipment I use.

4) I have no idea - but it has been at least a year at the shortest.

The first time this sort of contamination popped up was in Decemeber of 2010 - and it occured while some construction was being done that directly effected the lab in which my materials are stored (which is different from the lab in which the hoods are in). This contamination was cleared up easily after switching to a fresh bottle of FBS so I'm hesitant to say the contamination had anything to do with the construction. However, that construction finished up in the first week of January, and I haven't had another problem with contamination until recently.

Thanks for the help!

#109975 Help ID the contamination

Posted by dw237606 on 16 May 2011 - 10:50 AM in Tissue and Cell Culture

I've had this same looking contamination pop up twice prior to this time. I went on vacation for a little over a week and when I came back today and looked at my cells (A549 cells in DMEM + 10% FBS, and J774.A1 macrophages in DMEM + 10% FBS) this is what I saw (I had a lab tech do the subculturing for me while I was away).

Can anyone help me identify this contaminate and what the proper steps would be to get rid of it? I have already tossed out all of the flasks, cleaned the incubator, and prepared and filtered all new media solutions.

EDIT: I forgot to mention that the media was turbid, with very obvious white "dots" stuck to the bottom of the flask.

Attached Thumbnails

  • a549.jpg

#108675 Fungal Contamination

Posted by dw237606 on 03 May 2011 - 07:31 AM in Tissue and Cell Culture

Alright, that is what I was thinking. Thank you for confirming it. I disposed of the cells and am going to clean the incubator and everything else before starting them up again.

#108603 Fungal Contamination

Posted by dw237606 on 02 May 2011 - 10:45 AM in Tissue and Cell Culture

I have a flask of cells A549 lung cancer cells in DMEM + 10% FBS that we have been exposing to a novel anti-cancer agent for the last 3 months at very low levels in order to see if we can get a cell line to become resistant to the new drug and then look at gene expression to see how it differs from cells that arent resistant to the anti-cancer agent. However, starting last week ( 6 days ago) the flask has become contaminated with what Im thinking is either yeast or fungus. The medium is slightly turbid, with no noticeable color change from normal. Over the weekend the contamination grew to the point where visible white specks can be seen hanging in suspension in the medium. Furthermore, treating with pen/strep caused no change in the contamination, neither did gentamycin nor ampho-B all at their recommended concentrations.

When the contamination was first noted it was at a low enough level that it wasnt interfering with cellular proliferation and as long as the cells were washed and sub-cultured every day it was manageable. However, over the weekend the contamination has increased significantly and the cells are starting to lose their morphology. A small white speck began to grow on the top portion of the culture flask giving me further evidence that this is most likely caused by a fungus.

Im at a total loss as for what to do now since nothing seems to be helping clear it up. I would rather leave discarding the culture entirely and starting over as an absolute last resort since that will delay us 2-3 months but it looks like that might be the only option.

#106011 Xenograph - how to maintain cell viability?

Posted by dw237606 on 06 April 2011 - 05:35 PM in Tissue and Cell Culture

I am going to be preforming a xenograft on rats in order to develop a brain tumor. However, I am unsure as to the best way to maintain the cells - alive and healthy - during the 45-60 minutes it takes to preform the surgery. Will the viability be severely affected if I leave the cells (RG2-an adherent cell line)in suspension in a 15 mL tube in their growth media (DMEM + 10% FBS) outside of the incubator for approximately 1 hour?

As always thank you in advance for your help!

#103609 Precipitate forming after MTT !

Posted by dw237606 on 14 March 2011 - 04:18 PM in Tissue and Cell Culture

The media for your cells - what % FBS is it (assuming it is supplemented with some amount of FBS)?

When you say "I then tried the SDS/HCl solution and it never turned purple !!! just yellow !!" do you mean that after the MTT solution was removed, and the cells/remaining dye were resolubilized in the isopropanol or SDS solution, the solution turned yellow? Or are you referring to the yellow color of the solution of MTT added to the cells prior to the solubilization in DMSO/isopropanol/SDS?

How long are you incubating the MTT dye in with the cells prior to attempting to solubilze it in SDS/etc?

#102746 Centrifugal concentrators

Posted by dw237606 on 04 March 2011 - 05:06 PM in General Lab Techniques

These filtration units are a touch on the expensive side, and while I don't use them for proteins, my colleagues that use them for protein work, clean and reuse the tubes a few times before tossing them out.

#102744 Precipitate forming after MTT !

Posted by dw237606 on 04 March 2011 - 04:35 PM in Tissue and Cell Culture

Have you tried using DMSO instead of isopropanol?

What is the pH of the isopropanol you are using?

#102309 Yet another contamination question

Posted by dw237606 on 01 March 2011 - 08:40 AM in Tissue and Cell Culture

I can't give you any answers as to what it is, but I can say that I have seen something very similar when preforming cell counts, however, when I see these fibers it's never been more than 1 or 2 on the entire hemocytometer.

#102212 CTLL-2 culture - preventing cell clumping

Posted by dw237606 on 28 February 2011 - 12:28 PM in Tissue and Cell Culture

Gently swirl the plate when finished subculturing prior to placing it back in the incubator. While this is anything but perfect it does help quiet a bit. It's worth a shot.

#102211 mtt at early time points?

Posted by dw237606 on 28 February 2011 - 12:25 PM in Tissue and Cell Culture

I plate my cells 12-24 hours before changing the media out and adding my various treatments.

#101448 Trypsin for J774A Macrophages?

Posted by dw237606 on 21 February 2011 - 01:52 PM in Tissue and Cell Culture

Can J774A mouse macrophage cells be trypsinized for subculturing or would it be better to scrape them? Currently I'm scraping but would like to use trypsin if possible. Thanks.

#95765 Repeat bacterial infections

Posted by dw237606 on 22 December 2010 - 11:08 AM in Tissue and Cell Culture

I will try to subculture the bacteria, but that will be challening since the school is shutting down for the next week or so. If I'm able to do so, I will report back with the ID asap. Thanks.

#95590 Repeat bacterial infections

Posted by dw237606 on 21 December 2010 - 06:28 AM in Tissue and Cell Culture

Thank you for you responces. I will give the gentamycin a try. What percentage do you recommend?

The thing I find odd, but possibly unrepeated, is I've never had a problem with bacterial infections before. Then 2 weeks ago the cells devloped a bacterial infection and died. Thats when I began to thaw out new vials, and these too developed bacterial infections. Is there possibly a link between the original infection 2 weeks ago and these repeat infections?

When I froze the stock down the cells were healthy and free of any signs of a bacterial infection. Furtheremore, I thawed out 1 vial each time I froze down several vials to check the viability, and had no problems with bacterial infections with these cells in the weeks after thawing.

#95465 Repeat bacterial infections

Posted by dw237606 on 20 December 2010 - 06:11 AM in Tissue and Cell Culture

It started last week when I came in and all of my cells (A549) had a bad bacterial infection that killled them.

Media is DMEM, 10% FBS, 2% strep/pen

I thawed out a new vial, looked fine for 2 days, then they developed a bacterial infection and died.

Thawed out a second vial, looked good for 3 days, then they devloped a bacterial infection over the weekend and died.

I cannot find the source of the repeat infections. I used brand new media, brand new cell flasks, cell flaks from a different manufactor, put the cell flask in a different spot in the incubator, and ramped up the pen/strep to 5%. Yet, still my cells got a bacterial infection and died while all the other cultures in the incubator are doing fine.

What in the world can be causing these infections? I liberally spray all materials and the area in the fume hood that I'll be working in with 75% ethanol as well as leave the UV light on in the hood every time I finish. Help?

#95121 How much trypsin/Cause of death

Posted by dw237606 on 16 December 2010 - 08:49 AM in Tissue and Cell Culture

Well, I didn't add any base to my media that I know of. FBS and strep/pen aren't basic, and I went ahead and verified this with a tritration yesterday. I titrated the media that I had used, FBS, strep/pen, as well as original media, and the media from the dead cells. The media from the dead cell flask had a pH of 9.7 and the original media that was put in had a pH of 7.9. Are these the numbers that one would expect to find? (I only titrated each sample once, so I don't have the proper stats for these.)

The cells that were alive in the 96 well plate were successfully transfered back to a new culture flash. When I left last night at 5pm they looked to be in good health and had adhered to the flask. When I got in today, they were all dead and floating, and once again the media was a hue of purple.

So I had no choice today but to thaw out a cryovial. I mixed up NEW media, in order to rule out the possibility that the media itself was what causing the problem/contaminated. I'll report back with the results of this tomorrow.

Any ideas as to what could have caused them to become contaminated in the first place, and why cells that were healthy the day before would all be dead and the media so basic? Our stock of these cells is very small, only 2 more cryovials remain. I have been freezing down 2 or 3 vials worth per week, and this problem hit me before I could build up a substantial enough stock, so I really can't afford to kill the few remaining cells.

Thanks for all your help!

#94973 How much trypsin/Cause of death

Posted by dw237606 on 15 December 2010 - 07:19 AM in Tissue and Cell Culture

I'm not sure it was over growth or a bacterial infection that killed my A459 cells, but they're all dead. The media turned a shade that was slightly purple-ish (DMEM, 10% FBS, 1% strep). All of the cells in the flasks died, as well as 3 of the 4 96 well plates that I had plated yesterday, and the cells looked perfectly normal yesterday. So I'm trying to rule out all possible causes for their death.

Based on the color of the media, and the fact that all other cell flaks in the incubator survived without problems (CO2 didn't run out etc), I'm thinking possible over growth, but it seems very unlikely based on the number of cells I had yesterday.

Is 1% strep enough? Or should I modify it? In the past 1% has done the job without issue...?

Bacterial infection? Under the microscope there are a few long stringy (very scientific wording I know) items in there with the dead and detached cells. The morphology of the dead cells looks fairly normal, a little different but not much, except that they aren't attached to the bottom of the flask, but are in large "rafts" so to speak.

Possibly too rough centrifuge? The centrifuge I always used in the past is broken, so I used a different centrifuge. I normally spin them at 1500 rpms for 5 mins, this time I spun them at 1100 rpms for 8 mins. (1100 rpms for 5 minutes left a large amount of the cells still in suspension so I did it for another 3 minutes) But this is unlikely, since I have 1 plate that survived and none of the others survived.

Myo tested negative.

Whatever it was that killed the cells, I have one 96 well plate that survived, so I want to transfer the cells in the wells in this plate back into a culture flask. How much trypsin should I add, just enough to cover the bottom of the wells? When doing subculturing I use 5mL in the culture flask.

Thank you very much for your help. I know I have a lot of silly questions, but please bear with me (and talk slowly) as I am a chemist by training and have never done any cell culture work in the past. Baptism by fire?

#94102 Enough media?

Posted by dw237606 on 06 December 2010 - 10:58 AM in Tissue and Cell Culture

I'm going to be doing a few MTTs, in which my treatment incubates with the cells (10,000/well) for 24/48/72 hours. Is 100uL of complete growth media enough to keep that number of cells alive for the required 72 hours?

#91501 Cell Uptake Study

Posted by dw237606 on 05 November 2010 - 04:13 PM in Tissue and Cell Culture

I've been using Triton X-100 to lyse the cells before fluorescence detection, and while it has been working, it isn't the easiest to work with due to the excessive amount of bubbles it creates as well as it's viscosity. Both of these factors make it very challenging to deliever a consistent amount to each well, or to remove a consistent amount from the wells to use for a protein assay. Other than warming up it up, is there anything else that will make it easier to work with? I've been using it in a 10:1 solution with PBS (300uL of PBS with 30uL Triton into each well of the 24 well plate), is this dilution making the situation worse?

Thanks again!

#90732 Problem with Thawing (Freezing)

Posted by dw237606 on 28 October 2010 - 06:58 AM in Tissue and Cell Culture

The eye cells do require some additives to the media, exactly what and how much I can't remember off the top of my head. The media I used when attempting to thaw them did contain all of the required additives.

Thank you for confirming that the DMSO is removed in the spin. I changed the media 18-22 hours later just to be absolutely sure that all the DMSO had been removed. It helps me sleep at night :-)

#90663 Problem with Thawing (Freezing)

Posted by dw237606 on 27 October 2010 - 12:05 PM in Tissue and Cell Culture

I'm having a problem when thawing out cells from liquid nitrogen. I am trying to thaw 3 cell lines: A549 (lung cancer), RG2 (rat glioma) and human eye cells. TO start with none of the cells were frozen by me. The eye cells were frozen by one of the immunology doctors. Likewise the RG-2 cells were frozen by one of the pharmacists, and the A549's were frozen by a grad student who has since graduated and is now long gone. When I thawed the eye cells out, not a single eye cell survived, a fair amount of the RG-2 cells survive, and only a handful of the A549 cells survived. I'm concerned about the A549 cells, since I only have 1 more vial of these cells, the others I have about a dozen.

The protocol for thawing that I was given to follow went as follows:

Keep the cryovial on ice, and quickly add 500uL of warm media to the frozen cells. Pipette up and down a few times and then transfer 1000uL of the contents to a 14mL centrifuge tube containing 10mL of warm media. Repeat this process until the contents of the cryovial are empty. Then spin the tube for 5 mins at 1.5g. Aspirate the supernautant, and resuspend the pellet of cells in 1000uL of fresh media. Transfer this media into a culture flask containing 20mL of warm media.

I had the tube spinning in a little under 3 minutes, so I would venture a guess that I satisfied the "quick" criteria. This is the same speed/lenght that we use when culturing cells, and it's been working without incident. And yet somehow the survival rates for the eye and A549 cells were none and a few dozen, respectively. This protocol worked without any real problems for the RG-2 cells.

The freeze media contains DMSO, but how much I don't know exactly.

To get to the point, I've got a handful of A549 cells being cultured at the moment, and 1 more vial still in the nitrogen. I would like to start doing cell uptake experiments as well as MTT's next week, so anything you much more experienced scientists can tell me to help more cells survive thawing (assuming the freezing procedure went properly) would be greatly appreciated. Thank you!

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