BioForum Method Discussion

cell plating density

Fri, 10 Feb 2012 21:04:33 +0000

hi everyone,
I am kind of confused with the number of cells needed to plate in a well plate..
data i have:
cell/ mL : 6.175 X 10^5
suspension volume (Trypsin + FBS) : 4 mL
I need to add 100 micro L of 10 ^ 5 cell/mL for the above.. how do i do that?
or
If i need to add 5 X 10^4 cells per well in a well plate..how do i do it?

please respond ASAP

Regards.

What kind of cell culture contamination is it?

Fri, 10 Feb 2012 18:19:34 +0000

Hi guys,

I need help to identify what kind of contamination happened to my cell culture. I have HEK293 just thawed from liquid nitrogen and split just once. Then I found this contamination:

When I swirled around the culture plate, a puff of powder-like or dust-like things were blew up from the bottom of culture plate and then sink down. It could be observed directly by eyes. This cell culture contaminator feels and looks a lot like the bacteria culture flask: when you swirl the bacterial culture flask, the cultured bacteria are blasted up.

1) medium is not turbid, still clear and with normal red or purple color (PH normal). (so , it is not bacteria contamination?)
2) cells are still attached well. no obvious cell debris floating in the medium. Checking under light microscopy, cells have normal morphology.
3) The contaminators did not have the shapes of thread, rod or branches. Anti-fungus agent not help either.

So, anyone has any ideas? Many thanks for your time.

USP 51 and Microbial Recovery Incubation Time

Fri, 10 Feb 2012 16:46:46 +0000

Well, I'm looking to base a test method on USP 51 Antimicrobial Effectiveness Testing and I'm having trouble understanding what the USP is intending. Anyone done this before? I'll copy the relevant section:

PREPARATION OF INOCULUM

Preparatory to the test, inoculate the surface of a suitable volume of solid agar medium from a recently revived stock culture of each of the specified microorganisms. The culture conditions for the inoculum culture are described in Table 2 in which the suitable media are Soybean–Casein Digest or Sabouraud Dextrose Agar Medium .
To harvest the bacterial and C. albicans cultures, use sterile saline TS, washing the surface growth, collecting it in a suitable vessel, and adding sufficient sterile saline TS to obtain a microbial count of about 1 × 10⁸ colony-forming units (cfu) per mL. To harvest the cells of A. niger, use sterile saline TS containing 0.05% of polysorbate 80, and add sufficient sterile saline TS to obtain a count of about 1 × 10⁸ cfu per mL.
Alternatively, the stock culture organisms may be grown in a suitable liquid medium (i.e., Soybean–Casein Digest Broth or Sabouraud Dextrose Broth) and the cells harvested by centrifugation, then washed and resuspended in sterile saline TS to obtain a microbial count of about 1 × 10⁸ cfu per mL. [Note—The estimate of inoculum concentration may be performed by turbidimetric measurements for the challenge microorganisms. Refrigerate the suspension if it is not used within 2 hours. ]
Determine the number of cfu per mL in each suspension, using the conditions of media and microbial recovery incubation times listed in Table 2 to confirm the initial cfu per mL estimate. This value serves to calibrate the size of inoculum used in the test. The bacterial and yeast suspensions are to be used within 24 hours of harvest, but the fungal preparation may be stored under refrigeration for up to 7 days.

The table 2 they reference has a column for incubation temp, inoculum incubation time and microbial recovery time. This is what's throwing me off.
Way I see it, you get a starting population of 10^8 CFU's based on turbidometric measurements (and if anyone knows of a good chart to show this it'd be appreciated) and then you...what? Where do the incubation and recovery times come into play? They say to use the suspensions within 24 hours. How do you use these times to determine the population? I'd just take a sample and do a membrane filtration test on it to get the population, run concurrently with the actual inoculated samples.
Anyone run this before or understand what they are talking about with these "inoculum incubation time" and "microbial recovery times?" I suspect they are making it more complicated than it is.
First time post, hope to scan the archives soon to glean some good info

How to interpret this agarose gel?

Fri, 10 Feb 2012 15:37:57 +0000

Hello everybody,

Someone could help me to interpret this gel?
I did a PCR to confirm the efficacy of my recently designed primer. The primer must to product a 127pb amplicon. I see a band with this size in the samples 4 in both kits. But something appears below 100pb. What is this? Why my primer works in sample 4 and in the others not?

Some sugestions?

Attached thumbnail(s)

western blot loading control

Fri, 10 Feb 2012 14:04:50 +0000

When I am using loading control antibodies in my western blot (I am suing CDC-42 and GAPDH) I see multiple bands. CDC 42 has the MW of 21 but I see a very strong band at 43 kDA and more some not soo strong inespecific bands. GAPDH I also see inespecific bands. Can be because I am not using a protease inhibitor when I am extracting proteins. I am extracting proteins from Candida albicans. It is diffcult to fing a loading control. I found tubulin, the signal was perfect, no unespecific bands, but I am working with Heat Shock Proteins, so Tubulin is also activate tubulin, so I cannot use tubulin to control teh loading of my proteins. with beat actin I got a very weak signal and a very high background. Wating for an answer, please.

Viability assay with propidium Iodide and Alamar Blue

Fri, 10 Feb 2012 13:39:38 +0000

Hi,

I performed cell viabilitty assay using Propidium iodide and Alamar Blue. The experiment was performed using TECAN microplate reader.
The results I got as a RFU (relative fluorescence units). Now I want to calculate EC50, but I don't know how. I do not want use any software, can I use Excel? Does 4 different concentration is enough to calculate EC50? should this concentration be converted to log.?
And what about RFU values (axis -y), should I convert them as well?

I would be very grateful for any help.

Cheers,

Magda.

Problem with preparing unicellular suspension from MCF7 cells

Fri, 10 Feb 2012 12:36:24 +0000

Hi there,
I have a big problem with preparing a unicellular suspension from MCF7 breast cancer cell line. I need to do this because I need to establish a SCID model of human breast cancer injecting the mice with exact number of cells.
The problem is that after I take off the cells from the flask (I have tried different things: Trypsin, Acutase and also a scrap tool), centrifuge and resuspend them I can't count them because cells form floating islands and groups, which doesn't allow correct counting. The same is happening when I am using C-26 colon cancer cell line.
If anyone knows how to solve this problem please do help me-this stops my work from the very begining!

low pcr product

Fri, 10 Feb 2012 12:16:02 +0000

i am trying amplification of a gene from genomic DNA, as my one of the primer had secondary structure i used high annealing temp and now could get pcr product but is not enough so i tried re-amplifying the pcr product with the primers, but it is not giving the product at all, why could this happen? any suggestions to solve this problem?

Help. Lentivirus. Good titer but no transduction

Fri, 10 Feb 2012 11:41:32 +0000

Hello, I am working with lentivirus particles, when I do the transfection of the viral plasmid in Lenti-x cells I can see the fluorescence of the GFP whithout problems. I have made the titration by ELISA of the protein p24 and the titer es 1.2*10E10 with a p24 lentivirus titer of Clontech. My problem is when I transduct neurons from primary culture o cell lines like lenti-x, hek293, or NSC34( motoneuron cell line) I can´t se the expression of the GFP.

Anybody can help me????????? Please I do not what can I do.

Determining the N- and C-terminal regions in a protein

Fri, 10 Feb 2012 09:48:01 +0000

Dear all,

So, something's been bugging me all night. I'm doing a literature review on the polyester synthase and a group of related proteins. Coming to the molecular aspect, I encounter the terms "N-terminal / C-terminal regions" often.

How do we (or in the near future, I) determine / decide where the N-terminal or C-terminal spans?

The first thought that came into my mind was a multiple alignment with the same protein in other organisms. But that’s only useful for those having a conserved N or C regions. What more, this particular group of protein has a “non-conserved N-terminal region” while the alpha/beta hydrolase fold domain is conserved in part of the C-terminal (see below).

1......98.............................................589
NNNNNCCCCCCCCCCa/bCCCCCCC

The regions based on the protein of the model organism. (conserved region in blue)

Until now, only the protein in the model organism is well-studied and annotated. So, another way is to align the amino acid sequence from the model together with those from other organisms. Where the C-terminal begins (aspartate 99), that position will be a benchmark of sort to the other query sequence. Still, I felt this isn’t very convincing.

Waitiing for some feedbacks / suggestions / pointers regarding this.

Thank you

DNA Shuffling

Fri, 10 Feb 2012 09:30:56 +0000

Hi,

I'm working since last month with a DNA shuffling strategy to improve the enzimatic activity of 1 target enzyme.

I follow the original protocol developed from Stemmer in 1994, but I have many problems after Primerless PCR.

I always get an amazing smear of big size. I use different dilutions of this product to performe the second PCR but I've never got the right size band (2.300 kb).

Does anybody have experience with this technique? May you give me some advice?

Thanks,
RV

RA-differentiation of SH-SY5Y

Fri, 10 Feb 2012 09:13:01 +0000

Hi,

thank you for this very helpful forum!

I am new to the differentiation treatment of cells and I am experiencing difficulties in achieving differentiation of cells. Any help is very much appreciated.

I want to differentiate SH-SY5Y cells (bought from ECACC via Sigma) with retinoic acid (R2625, Sigma Aldrich). For what I have seen in the literature, differentation should be achieved by treatment of cells with 10µM RA in full medium (DMEM, high glucose, L-Glutamine, non-essentail amino acids, 10% FCS or DMEM/F-12 with the same additives) for some time (3-7 days) with periodic media exchange every 2-3 days.

To my understanding, differentiated cells should stop proliferating and in case of RA on SH-SY5Y the cells should also show neurites. However, my cells simple continue to proliferate (just a bit slower than cells that only see DMSO but still fast) and I cannot see any difference in shape between vehicle-treated (DMSO) and RA-treated cells. I also tried higher RA-concentrations up to 100µM without success.

I solubilised RA in DMSO at 15mg/ml, sterile filtered it and store it at -80°C. When I add it to the medium I see that it immediately forms insoluble crystals that slowly sink to the bottom. I then vortex the mixture until I cannot see any crystals anymore.

Do you use FCS (FBS) in the differentiating media? If yes, how much? I started to think that maybe the growth-promoting effect of the FCS counteracts the RA although I have read that others use up to 15% FCS.

How do you prepare your RA? What do you do about the crystal formation?

Again, help is very much appreciated!

Thank you in advance
Johannes

Seeking advice Regarding contamination

Fri, 10 Feb 2012 06:29:22 +0000

Dear Guys ,

At first I am very gratef ul for your comments and advices.
I have no one except you to seek for your priceless help.
I need your advice in two issues:

1) I am in a middle of experiment using keratinocyte from ATCC, I cultured it firstly with Antibiotic then without antibiotic.
it was fine and I was doing my experiment on 24 well plate, it takes around 4 days.
the first, second and thirds day was ok, on the third day I change the medium and add new medium, and on the fourth day I saw something like sever white turbidity in 17 wells out of 24, and I cant see my cells, they are gone, not attached to the surface of my plate, nothing only in 24 hrs.
I have added the new medium using the same pippte and the same tip for all wells, and the reagents for my experiment too.
The most strange things is in another cultured 98 well plate (3 out of 98) showed the same sever turbidity, this was cultured in another day.
the rest of my plate was fine too, no turbidity and cells looks fine under microscope.
So I have took some photo using 100 X (oil immersion) as these intruders are very tiny and cant be clearly seen under 40X.

2) I am using new FBS, on culturing another type of cells (No Antibiotics too), and what I noticed that there are a lot of things floating in my medium under microscope, but not turbidity which could be seen by naked eye, I dont know if they are cell debris or debris from my new FBS itself.
so my question, to identify contamination what shall I expect ( I am not using antibiotics),
Sever turbidity??? or slight one.
Contamination occupy all my plate or minor one??
death of my cells and the first issue ( but my cells are 97% viable using trypan blue.)
I have added a photo from American National Cancer institute which describes what you can expect in contamination, A is non contaminated cultured cells, so shall I expect something like that and my cells be normal.

Thank you guys in Advance,
Best Regards

antibiotic assay

Fri, 10 Feb 2012 02:12:17 +0000

Anyone who is familiar with Gramicidin (antibiotic) assay? I have been performing this test for quite a while, but I could not come up with a valid result. I am using USP as my reference for the procedure (Spectrophotometry). The problem is, the turbidity of my assay do not have a trend even if I increase the concentration of the antibiotic used for the Standard. I am working with a pure culture of Enterococcus hirae, and all the materials used was sterilized, so I am confident that my problem with turbidity is not caused by contamination. Any insight will be highly appreciated. Thanks in advance.

xenograft cells harvested from spleen of SCID/NOD mice

Fri, 10 Feb 2012 01:04:54 +0000

Hi,

I'm use to working with cell lines and this is the first time I am working with xenograft cells harvested from the spleen of SCID/NOD mice. I am currently practicing an Alamar blue cytotoxic assay. I bring the cells up from liquid nitrogen and plate them the afternoon before I intend to incubate them with cytotoxic drugs for 48hrs and then adding the Alamar blue and measure the florescence at 0hr and 6hrs. This is a well established protocol by the lab I am currently in. My problem is that the cells in all of my wells (including controls) die before the day when I'm suppose to add the Alamar blue. I think that maybe its my freeze thaw technique that maybe causing the cells to die very quickly. The only criticisms I am getting is that I am really gentle when I bring the cells up from liquid nitrogen. Can someone please give me some tips on how I could be bringing the cells up successfully from liquid nitrogen or does anyone have any insight on what might be the problem??

Thanks.

Nickel Column Purficiation

Thu, 09 Feb 2012 20:36:29 +0000

We are having problems with the Imidazole that we use for elution in our Nickel Column. We believe that the Imidazole is tearing up our protein. Is there anything else I can use to help elute the Protein, instead of using Imidazole? BTW our protein has a 6X-His tag.

Weight of a single Lambda DNA.

Thu, 09 Feb 2012 20:11:51 +0000

Can anyone please tell me the weight of a single lambda-DNA molecule of 24000 bp?

How to prepare 1M EDTA with only Tris Base?

Thu, 09 Feb 2012 19:46:36 +0000

Hi, I've got a question:

How do I prepare 1M EDTA, pH 7.4? Only Tris Base is allowed for adjusting the pH, no HCl and NaOH can be added.

Is it even possible to do this? Because I can't seem to find a way.

Thanks for any help.

RBS site removed in construct. Will there be expression?

Thu, 09 Feb 2012 18:37:19 +0000

I inserted a gene of interest into a pET32a vector downstream of the T7 promoter. However, while doing this I also removed the RBS unintentionally. I plan to express the construct in BL21 E. coli to purify the protein of interest. My question is... will there be expression of the protein despite the lack of the RBS? Will the ribosomes be able to translate the mRNA without it? I am doubtful it can, but I am certainly trying to prevent having to make a new construct.

Thanks

Sarkosyl and Ni-NTA

Thu, 09 Feb 2012 17:33:26 +0000

Hi all,

I work with a 50KDa his tagged protein (cloned in pET 28 and pET Sumo) that is overexpressed in Rosetta cells. I have a solubility and purification problem.The protein is only soluble (partially) in the presence of 0.3% of Sarkosyl (I've tried different concentrations of IPTG, different temperatures, and different pHs in the lysis buffer). However, with sarkosyl the protein does not bind to Ni-NTA. I've tried to dialyze the lysate but it didnt work. I' ve heard about adding triton and chaps to the lysis buffer to make the protein bind to the colunm in the presence of Sarkosyl. The protein bound indeed, but then it doesnt come out, not even with the addition of EDTA! I already tried to express the protein in pMAL vector, but I had the same problem.

I'm expressing the protein with 1mM IPTG for 3 hours at 37C. My lysis buffer: 50mM Tris-HCl pH 7, 500mM NaCl,0.3% Sarkosyl and protease inhibtors, then I sonicate the cells.

Any of you gurs have an idea of what can I do to purify the protein? I will need the protein for crystallization assays.

Thanks in advance!

Teka