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jakatta70's Content

There have been 31 items by jakatta70 (Search limited from 21-February 19)



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#77434 THP-1 Cells differentiation

Posted by jakatta70 on 29 June 2010 - 12:45 AM in Tissue and Cell Culture

Hi,

I worked with THP-1 macrophages for a couple of years and never used poly-d-lysine coated plates. I simply used the conventional 12 well plates without any problems. You also mentioend that you were told to induce the macrophage phenotype by hitting them with LPS-IFNg combination. This would induce a classically activated phenotype and not a naive phenotype. If you wanted to induce a naive type macrophage hit 250,000 cells/ml with 20 nM PMA for 2-3 days.



#75960 polarized cells

Posted by jakatta70 on 18 June 2010 - 01:30 AM in Tissue and Cell Culture

Hi again,

Ignore my last post. I'm incorrect!

Here is a good review on the subject:

http://www.nature.co...rity/index.html



#75959 polarized cells

Posted by jakatta70 on 18 June 2010 - 01:26 AM in Tissue and Cell Culture

Hi,

In terms of macrophages if a macrophage has been matured with, say, LPS+IFNg, then it becomes POLARIZED to a classically activated state ie it will respond by producing TH1 cytokines in response to appropriate stimulus. If the macrophage is matured with TH2 cytokines, such as IL-4 + IL-13, it becomes POLARIZED to an alternatively activated state ie responds to stimulus with TH2 cytokines. Also unique markers for each polarized state will decrease or increase depending on what sort of macrophage you have polarized the initial macrophage to.

I don't think polarization has much to do with morphology although. I wouldn't think so. If a monocyte matures to a macrophage I would think that would be CONVERSION. I think POLARIZATION is more complicated and intricate.

I hope this helps you a bit.



#75470 Accidental thawing of frozen cells

Posted by jakatta70 on 15 June 2010 - 10:08 AM in Tissue and Cell Culture

Hi,

I dont know if moving them to -20C would damage them. However our lab recently had to move a good amount of cells from liquid nitrogen storage to -80C for 24 hours as our N2 tank had run out. These cells were able to grow no problem when thawed and cultured. The temperature shift was from -200C to -80 and the cells were ok. I would say your cells will be fine if they were left in -20C for only a short time period. Theres only one way to find out and thats to grow them though.



#75360 mononuclear cells medium

Posted by jakatta70 on 14 June 2010 - 12:44 PM in Tissue and Cell Culture

Hi,

I have just made up a sterilised solution of 200 mM (0.2 M) beta mercaptoethanol in the past and added that to my culture media in a sterile biological safety cabinet.

Use the equation of Concentration 1 multiplied by Volume 1 = Concentration 2 multiplied by Volume 2

Say you will have the standard 500 ml of culture media. You know the final concentration of mercaptoethanol is 50 uM and the starting concentration is 200 mM. 1000uM = 1 mM. Therefore 200 multipled by 1000 = 200,000uM.

So:

200,000 uM x V1 (in ml) = 50 uM x 500 ml

V1 = 25,000/200,000

V1 = 125 ul of mercaptoethanol



#71768 THP-1 Cells

Posted by jakatta70 on 20 May 2010 - 12:15 AM in Tissue and Cell Culture

Hi Bryan,

I used to run similar experiments to you with the macrophages. I was looking at classically and alternatively activated states using either cytokines or parasitic helminth extracts.

Yes you should be able to add the RLT buffer directly to the wells of the plate. The cells should burst open and you can then pipette the supernatant into a pre-chilled sterile tube on ice. Do everything in this extraction process on ice to slow down RNA degradation.

I thought I had a protocol saved for detaching THP-1 cells but I don't (only one for detaching Caco-2 intestinal cells). However the protocol might be similar to that used for intestinal cell lines (trypsin edta-pbs). If you check online or on NCBI you should be able to find a protocol easily.



#71682 THP-1 Cells

Posted by jakatta70 on 19 May 2010 - 12:07 PM in Tissue and Cell Culture

Hi Bryan,

When I used to extract RNA from THP-1 macrophages in 12 well plates I never detached them from the plastic. I simply added 1 ml of TRIzol (Invitrogen) and left at 4 degrees celsius for 10 min. I then extracted the RNA either the old fashioned way using chloroform, ethanol etc or by using the RiboPure kit from Ambion (I recommend this kit because of its ease of use and rapid, problem free process). I obtained yields of up to 200 ng/ul and 2.1 purity (NanoDrop spec) from 250,000 cells/ml. You might think this is low but all I needed was enough to make a solution of RNA at 10 ng/ul for qPCR purposes.

Give this method a try and see if it improves your results. I believ you onyl need to detach the cells carefully if you want to use them for things like Flow Cytometry.



#67410 labeling drugs with FITC

Posted by jakatta70 on 20 April 2010 - 05:08 AM in General Lab Techniques

Hi,

I work with indane drugs which have aromatic rings attached to various groups such as NO2, CN, F, CL etc etc. Id like to investigate how the drugs enter the cells Im using (Caco-2 and THP-1) using fluorescent microscopy; however I need to label the drugs with a fluorophor such as FITC.

Has anyone experience in this area and if so can you tell me if it is a standard and relatively straightforward procedure. Or is it one requiring a lot of money and submission of the drug to a company to attach the tag.

Many thanks.



#65534 Help with THP-1 cells

Posted by jakatta70 on 06 April 2010 - 11:15 AM in Tissue and Cell Culture

Hi,

I used to work with THP-1 monocytes in my first postdoc and I was interested in differentiating to classically and alternatively activated macrophages. I tried both PMA for 3 days and then specific cytokines thereafter as well as cytokines alone to induce distinct phenotypes. I found that cytokines alone was not enough to differentiate from monocytes to a specific macrophage phenotype. So Id advise PMA at 20nM for 3 days, remove media and then add your cytokines.

Im unsure about the mercaptoethanol question. Its function in media is to break down toxic metabolites from the cells. Id presume then that the small concentration of BME in your media wouldnt affect macrophage phenotype but I cant say for sure. You could contact Dr Judith Allen at the University of Edinburgh, Dr Sheila Donnelly at Brisbane or Dr Derek McKay at University of Calgary. They would easily be able to answer any questions on macrophages for you.



#65510 mouse macrophage cell line

Posted by jakatta70 on 06 April 2010 - 05:28 AM in Tissue and Cell Culture

You could also use J774 but I believe RAWs are easier to maintain.



#65509 flask or dish?

Posted by jakatta70 on 06 April 2010 - 05:26 AM in Tissue and Cell Culture

Hi,

I regularly use T75 flasks to grow my cell lines and then seed them into 12 well plates or 60 mm dishes for experiments. I then add 1 ml of TRIzol to each well of a 12 well plate. However I have also added 1-2 ml TRIzol to T25 flasks in the past and it works fine. Although it is standard practise to scrape the cells off the flask surface I have never needed to do this. Leave the flask+ TRIzol in the fridge for 10 min and your cells will have burst open.



#64553 Very spall particulates vibrating in my culture

Posted by jakatta70 on 28 March 2010 - 10:34 AM in Tissue and Cell Culture

Hi,

It wouldnt be Mycoplasma as you will not be able to see Mycoplasma. You'd need a PCR or some other method to detect Mycoplasma.

I have observed similar particles and also thought I had contamination. It turns out that they are just pieces of FBS or cell membrane components moving via Brownian motion. If your cells arent undergoing apoptosis, necrosis or lifting off the plates (or even taking up typan blue stain) then your culture are fine.



#63228 kind of contamination?

Posted by jakatta70 on 19 March 2010 - 12:42 AM in Tissue and Cell Culture

Hi,

I used to work with THP-1 monocytes/macrophages and I found that the media is "milky" when the cells are in suspension and at a high density. The media might turn milky if you had a bacterial infection in the flask but not sure.

I would throw out your media and the cells/flasks etc and take your time decontaminating the hood. Spray everything down with ethanol and a fungicide and it might be a good idea to have a more experienced lab member watch you perform cell culture and then provide a critique of it. Ive been doing cell culture for a few years now but I was in doubt recently about my aseptic technique and asked to be monitored briefly.


Where testing for Mycoplasma is concerned there are several commercially available means to use, each with their own advantages and disadvantages. I have attached a PDF document for you to look at. We use a standard PCR method but this can sometimes give you false positives. Other methods are both time consuming and expensive.

Attached Files




#62954 Contamination in cell cultures

Posted by jakatta70 on 17 March 2010 - 05:12 AM in Tissue and Cell Culture

Hi,

I completely concur with Clare. This is only a piece of plastic from the wrapping which either the pipettes or flasks etc have been in. I wouldn't worry about it. We see it regularly in our cell lines and it does not seem to have any disastrous effect on growth, apoptosis, necrosis etc.



#49298 Ethanol/methanol/dmso for cell culture

Posted by jakatta70 on 03 December 2009 - 06:04 AM in Tissue and Cell Culture

I am using tamoxifen on my macrophage cultures. As it is in powdered form, I will be using a solvent. What purity ethanol and methanol is safe for cell culture? I know for DMSO 0.1% is the best - I am using that for other inhibitors so I also have a dmso vehicle control to be safe. Are ethanol and methanol toxic to cells? What should I dilute with?

Thanks


I carried out an LDH assay for a range of ethanol and methanol concentrations and found that I could go as high as 2% in both without significant adverse side effects on cell viability. This is using Caco-2 intestinal cells so I don't know if you would find the same for macrophages. I think you'd be better performing a cell death assay for a range of solvent concentrations to be on the safe side.



#48669 THP-1 culture protocol

Posted by jakatta70 on 01 December 2009 - 03:59 AM in Tissue and Cell Culture

Hi everyone,

I worked with the human monocyte line, THP-1, in my first postdoc and never had any problems with it. The only "problem" I had was having too many cells! Im now in a postdoc position elsewhere and I purchased THP-1s from ATCC. The first vial simply didn't grow and then the numbers of cells started to decrease significantly after every centrifugation (120 g for 5 min) presumably because the dead cells floated away into the media. Although I was confident that the cells weren't dying because of my aseptic tenchnique, I purchased a new vial anyway. Again the cells have died!! Im very frustrated. :angry:

Here is the composition of my media: RPM1 1640 containing 2 mM glutamine; 10 mM HEPES; 1mM sodium pyruvate; 4.5g/L glucose (25 mM; can someone check that the molarity I have stated is correct for 4.5g/L glucose?); 1.5g/L sodium bicarbonate (18 mM; again can someone check?); 10% FCS; 0.05 mM beta mercaptoethanol.

I thawed the vial, cleaned it, transferred the cells to a 50 ml tube, added 9 ml of media dropwise, centrifuged at 120 g for 5 min; removed supernatant; resuspended in 1ml of media and seeded at 200,000 cells/ml in a T25 flask for 7 days, adding media every 3 days. Now I must say that this isnt the technique I have used previously. I used to add 30 ml of media to the cells and let them sit overnight at 37C before centrifuging them. In addition the media composition advised by ATCC is different than what I used before. I added 17.5 ul of 1M HEPES, 10 ml of penicillin streptomycin, 10% FCS. :huh:

Can anyone advise me on what Im doing wrong? Has anyone had similar problems with THP-1s from ATCC?

Many thanks. :lol:

Michael.



#42788 Stimulating/Drugging Mammilian Cells

Posted by jakatta70 on 05 November 2009 - 07:43 AM in Tissue and Cell Culture

Does anybody know the proper way to stimulate a mammalian cell culture with drugs?
I am using well plates, and am unsure if I should add the drug directly to each well containing medium (e.g., a 1:100 dilution of pre-diluted drug in DMSO to DMEM), or I have also read a proper way to do it is make a stock solution of drug in the medium using a 1:1000 drug:DMEM dilution.
If anyone has experience, please help. These are kinase inhibitors.



Hi,

If you are working with a chemical as toxic to cells as DMSO is, I would advise making a high concentration stock in DMSO and then bringing the concentration down, into aliquots, using your media of choice (DMEM). This will ensure your cells are exposed to as little as possible of DMSO.

Some people arent able to further dilute their drugs if they are only soluble in organic colvents like methanol. Im working on a drug which is soluble in methanol but when I add 10 microlitres into 990 microlitres of water, it quickley precipitates out.



#41455 MMP Zymography

Posted by jakatta70 on 28 October 2009 - 04:06 AM in SDS-PAGE and Western Blotting

Hi all,

Ive a quick question regarding MMP zymography. We incorporate gelatin into the gel and allow the MMPs in the loaded sample to digest the gelatin overnight.

After 15 hours Ive been used to seeing nice strong bands of MMP2 and some less strong bands of MMP9. We have recently bought in new gelatin and made a new solution. However now we dont see any bands! My supervisor assures me this is normal with new gelatin and he seems to be correct as he is able to see his bands on the gel after he stains in and destains it.

I just want to know what makes an "older" batch of gelatin better at visualising bands after 15 hours while "newer" gelatin doesnt reveal anything until the gel is stained.



#40772 DMSO and cellular culture

Posted by jakatta70 on 22 October 2009 - 02:10 PM in Tissue and Cell Culture

Hello

I have the following problem: I have to use a compound that dissolves only in DMSO 100%. I need to use it in cellular cultures of certain endothelial cells and stellate cells, that are exquisivitely sensitive to DMSO. At 0.01% DMSO, those cells die. I need to use the compound at 1 to 10 micromol/l concentrations, and I am not able to make a stock solution above 10 mmol/l, or this compound does not dissolve even in DMSO. So I can't go below 0.01% concentration of DMSO in my cells (am I clear or not at all ???)
has anybody an idea ?

Thanks a lot +++


Hi,

I experienced a similar problem recently. I performed a concentration range of DMSO with my Caco-2 cells and assessed for cell death via an LDH assay. I found that concentrations of 1%+ were toxic. So in order to get rounf this I made a 10 mM stock solution of my drug in 100% DMSO. I want t a final concentration of 10 uM that the cells are exposed to in 1ml of media. This equals 1 ul of stock solution to 1 ml of media which represents 0.1% DMSO.

Have you performed a cell death assay to confirm that your cell lines cant take anymore than 0.01% DMSO? Its just that the figure seems quite small to cause toxicity.

Perhaps make your stcok solution as concentrated as possible in 100% DMSO and then bring the concentration down by diluting in sterile water. Your drug should still remain in solution while reducing the final DMSO concentration that your cells are exposed to.



#38226 Drugs first and then cytokines, or at same time?

Posted by jakatta70 on 30 September 2009 - 03:00 AM in Tissue and Cell Culture

Hi,

I am investigating the immunosuppressive/anti-inflammatory effects of novel drugs using the Caco-2 cell line. The drugs are envisioned as novel therapeutics for Crohn's disease and Ulcerative colitis. We add TNFa and IL-1b to the cell media to induce the production of pro-inflammatory markers, such as MMP-9.

For a few months I have been adding the cytokines and drugs simultaenously. The drugs havent been reducing MMP-9 or other markers, even though I know that these two specific drugs worked prior to me starting this project. My supervisor has advised that I should be treating the cells with the drug, waiting an hour, and then adding the cytokines.

The problem I have here is that this doesnt make sense from a therapy viewpoint. If the drugs are only effect at blocking the initial pro-inflammatory signal but cant reduce an established signal, then should I be performing the experiment in the way advised by my supervisor?



#37478 antibiotic stability in culture medium

Posted by jakatta70 on 24 September 2009 - 07:00 AM in Tissue and Cell Culture

Hi,

Check out this weblink. It might be helpful to you. I personally think its overkill as I and other members of my lab open our media bottles in the safety cabinet every day and use in cell culture and we have had no problems with bacterial contamination etc as of yet.

http://www.meditechi...df/Medicult.pdf



#36592 Haemocytometer cell count

Posted by jakatta70 on 16 September 2009 - 08:23 AM in Tissue and Cell Culture

Im almost certain you may use any coverslip. I usually use a 0.2mm one.



#35749 Methanol toxicity in cell culture

Posted by jakatta70 on 09 September 2009 - 12:38 AM in Cell Biology

Good luck, sounds like it should work. I like your 'try it out and see' attitude. Are we allowed to know a bit more detail about your compounds?


Hi,

Im bound by a confidentiality agreement but I can say that they are extremely polar compounds with high molecular weights.

One of my supervisors (I have three unfortunately and two arent even biologists. They are chemists that havent a clue about the assays Im doing!) sent me a graph that a previous student had done with on drug in question. He had told me the drug reduces MMP-9 in vitro. I havent seen that and my n number is 3 while the other previous student only tried this assay once. Well, I get the graph yesterday and there is no MMP-9 reduction over a range of drug concentrations! lol! Maybe I should advise the supervisor to have his eyes (and head) examined!



#35704 Exploding cryovials

Posted by jakatta70 on 08 September 2009 - 01:03 PM in Tissue and Cell Culture

Hi,

I experienced a similiar problem when I first started working with frozen cell cryovials. Unfortunately the only thing I could suggest to you is to very quickly take your vial from storage and transfer it very quick to a biological safety cabinet and then unscrew the cryovial lid off to avoid pressure build up. This ought to stop the cap from exploding off and wasting the vial contents. Let me know if this works.



#35655 Methanol toxicity in cell culture

Posted by jakatta70 on 08 September 2009 - 03:36 AM in Cell Biology

Dissolving the drug into the serum is an interesting idea. I will certainly try that. Could you tell me though why you think this might keep the drug in solution?


If it weren’t for the carrier proteins in serum, my drugs would never get to where they are needed :)


Hi DRT,

I took your advice and tried dissolving some of my highly polar compounds into FCS. Unfortunately none would dissolve even after I applied 50-60C heat and/or vortexing for an hour. So I decided to try a 1:1 ratio of 100% DMSO + FCS. This produced an intense exothermic reaction which precipitated the protein content of FCS and turned the solution a dark cream colour. So I cant use this in my in vitro assays.

So what Im going to do is make a concentrated solution of my drugs in 100% DMSO and add x microlitres to my cell assay so that the final concentration of DMSO the cells are exposed to will be less than or equal to 0.5%. At this percent I havent been finding toxicity when using an LDH assay. Fingers crossed!




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