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Piersgb's Content

There have been 41 items by Piersgb (Search limited from 20-November 18)



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#94718 Lentivirus toxicity

Posted by Piersgb on 13 December 2010 - 04:40 AM in siRNA, microRNA and RNAi

Why not use the 0.22um filter?



#94715 Differentiation of CD11b cells into macrophages.Help please

Posted by Piersgb on 13 December 2010 - 04:15 AM in Immunology

GM-CSF and M-CSFare the two growth factors giving rise to monocyte/macrophage-restricted progeny.

http://nic.sav.sk/lo...fic/node21.html



#94714 Anitbiotic shelf life in liquid media at RT

Posted by Piersgb on 13 December 2010 - 03:54 AM in General Lab Techniques

Shelf life @ fridge temperature of main cell culture antibiotics:

http://www.bioxys.co...antibiotics.htm


When in tissue culture, half life of antibiotics is drastically reduced:

http://www.ncbi.nlm....o00231-0098.pdf
http://www.invitroge...timycotics.html



#90630 how to make Percoll solutions?!

Posted by Piersgb on 27 October 2010 - 06:30 AM in Hematology

I work with mouse bone marrow too! I've also been playing with Percoll and from studying the manual I've come up with the following:

You first need to make a 1.5M solution of NaCl. Then to 9 parts Percoll (from the bottle) you add 1 part 1.5M NaCl. This is to make Percoll isotonic, and from then on the manual refers to this solution as Stock Isotonic Percoll (SIP) as 100% Percoll (confusing, I know!).

To make further dilutions of Percoll, you dilute with 0.15M NaCl.

To get a 60% Percoll solution you want to have a final solution that is 60% SIP : 40% 0.15M NaCl. So to make 10mls of 60% Percoll, mix 6ml SIP with 4ml 0.15M NaCl.

Hope that helps!



#89336 Pointless masters

Posted by Piersgb on 12 October 2010 - 06:01 AM in Venting and Counseling

At whichever institution you're at, is there any code of conduct document/rules that you could refer to when it comes to unfair treatment? Maybe try talking to higher powers at your institution. Explain the situation, ask for witnesses to confirm what you've seen/felt.

Sometimes people aren't meant to teach because they act like your supervisor, and it's so unfair on the students. It does sounds like there have been so many internal failings for them to allow you to go all the way through to submission and then quote the quality as 'appalling'. That is awful.

What is your secondary supervisor like? Are you on good terms with them? Maybe explain the situation to them fully?



#88965 CD19 Expression at various stages of plasma cell differentation

Posted by Piersgb on 07 October 2010 - 03:33 AM in Immunology

Have a look at some of these links:

http://www.abdserote...ouse/index.html

http://www.biolegend...pfeatured&id=17

http://www.biolegend...on_Proteins.pdf

http://www.miltenyib...ouse_cells.aspx



#88411 Penicillin and Strep

Posted by Piersgb on 30 September 2010 - 03:04 AM in Cell Biology

You could just buy Pen-Strep 100x solutions from somewhere like Invitrogen. They're not very expensive at all! As these products are the standard used in cell culture, you wouldn't need to worry about validating how you made up your own Pen-Strep solutions. I think it could save you some time and effort..!

http://products.invi...=search-product



#87524 Immunofluorescence using dead cells?

Posted by Piersgb on 21 September 2010 - 07:35 AM in Immunology

I guess the problem with immunostaining of dead cells is that upon the initiation of apoptosis and/or necrosis the cells may undergo a lot of surface and intracellular membraneous protein changes. In other words, as soon as the cells start dying they may begin to shed the normal antigens that you would expect to get in the usual places.

So I guess you could try and catch these cells to as near as death as you can and then fix them with something like 4% Paraformaldehyde (PFA) in 1xPBS. This would preserve surface antigens. If you wanted to get to the intracellular stuff you could permeabilise them with a detergent such as Triton-X100. Not sure if this would help the PFA get in and fix the interior of the cell.

The other option I guess would be to try and find some antigens that continue to exist and thus allow you to stain after the cells die.

Just my two cents!



#87409 Culture Media for stem cells

Posted by Piersgb on 20 September 2010 - 05:54 AM in Stem Cell

Do you use any cytokines at all? How are you isolating the stem cells from the blood?

There are plenty of companies out there that specialise in stem cell media and reagents, I guess it may be case of trying a few out and seeing if they work for you.

StemCell Technologies are quite a big one, but there are many more out there! You may want to try a serum free media or if you do use serum use serum that has been specially batch tested for stem cell culture.

http://www.stemcell.com/



#87406 my cells love infection, any thoughts on antibiotics?

Posted by Piersgb on 20 September 2010 - 05:50 AM in Tissue and Cell Culture

Penn-step is used quite commonly in cell culture media. I use it in my culture with primary mouse bone marrow cells and have had very few obvious infections. I must say that I have never tested for mycoplasma infections, but if you're worried about this then perhaps you should run some tests on this?

So I guess I would recommend you try Penn-Strep for a bit and see how that goes. I use it at 100 IU/mL penicillin and 10 g/mL streptomycin. I don't think this would work against mycoplasma infections as they don't have cell walls. They are also very small and so you probably wouldn't be able to filter these out and you wouldn't see them under the microscope. There are commercial treatments available that can rid infections.



#86716 Murine antibodies

Posted by Piersgb on 13 September 2010 - 03:30 AM in Hematology

Yep, have had a good browse already. What I'm really after is an established group of antibodies that are used to identify as many of the different cells types in mouse blood/bone marrow. So ideally one antibody for each cell type (B cells, T cells, Lymphocytes, T-reg, Basophils, Neutrophils, Eosinophils, Stem cells).



#86702 Murine antibodies

Posted by Piersgb on 13 September 2010 - 02:35 AM in Immunology

I'm currently trying to compile a panel of antibodies that would generally identify individual blood cell types in murine peripheral blood and bone marrow. For example, I'm using a Siglec F antibody to identify murine eosinophils.

Does anybody else have any experience or know where I could find a good set of antibodies to label most/all murine blood cell types?

Any help would be greatly appreciated.



#86701 Murine antibodies

Posted by Piersgb on 13 September 2010 - 02:34 AM in Immunology Products

I'm currently trying to compile a panel of antibodies that would generally identify individual blood cell types in murine peripheral blood and bone marrow. For example, I'm using a Siglec F antibody to identify murine eosinophils.

Does anybody else have any experience or know where I could find a good set of antibodies to label most/all murine blood cell types?

Any help would be greatly appreciated.



#86700 Murine antibodies

Posted by Piersgb on 13 September 2010 - 02:32 AM in Hematology

I'm currently trying to compile a panel of antibodies that would generally identify individual blood cell types in murine peripheral blood and bone marrow. For example, I'm using a Siglec F antibody to identify murine eosinophils.

Does anybody else have any experience or know where I could find a good set of antibodies to label most/all murine blood cell types?

Any help would be greatly appreciated.



#86699 T helper differentiation and staining

Posted by Piersgb on 13 September 2010 - 02:26 AM in Immunology

Which detergent are you using and how much of it? It could be that the detergent that you are using is too strong and is destroying the cells? Have you done any fixation steps?



#86698 Dying endometrial stromal cells

Posted by Piersgb on 13 September 2010 - 02:24 AM in Stem Cell

Are you sure there is no contamination? Are you using any antibiotics in your culture medium?

You could also try changing the medium every 48 hours as they did in your articles. Do you stimulate them with any cytokines to keep them alive at all?



#86697 Is IgG monoclonal antibody better then IgM? why?

Posted by Piersgb on 13 September 2010 - 02:18 AM in Immunology

I'd assume that IgG monocolonals are more popular because, as you said, they are easier to generate and purify and then more stable to work with.

IgM monoclonals may have more binding sites but surely only one antibody can bind to one antigen on the cell surface due to spatial flexibility? I think the multiple binding sites of IgM would only really matter if you were using the antibody to neutralise a substance that was in solution.

Loads of companies produce antibodes and a large number of these are IgG antibodies. I'd take from this that they've tried and tested their methods for Ab production and have ended up favouring IgG Abs. Though when it comes to raising an Ab for your specific antigen it could be wise to try both!



#84943 Giemsa, Wright and May-Grunwald stains

Posted by Piersgb on 27 August 2010 - 01:43 AM in Histology and Pathology

Does anyone have much experience with these stains when it comes to murine granulocytes?

I'm looking at staining mouse bone marrow cells on slides and also cell culture preps of similar cells/granulocytes. I've been having problems getting the stains to do what they say they should. I'm beginning to appreciate that the pH of the stain and any wash steps is important for the type of cell you're interested in (eosinophilic/basophilic). I'm mainly looking for Eosinophils so have the pH at 6.6 at the moment and would love to able to identify their acidophilic granules and bilobed nuclei!

Does anyone have a protocol that they've used sucessfully here?



#84826 Giemsa, Wright and May-Grunwald stains

Posted by Piersgb on 26 August 2010 - 03:12 AM in General Lab Techniques

Does anyone have much experience with these stains when it comes to murine granulocytes?

I'm looking at staining mouse bone marrow cells on slides and also cell culture preps of similar cells/granulocytes. I've been having problems getting the stains to do what they say they should. I'm beginning to appreciate that the pH of the stain and any wash steps is important for the type of cell you're interested in (eosinophilic/basophilic). I'm mainly looking for Eosinophils so have the pH at 6.6 at the moment and would love to able to identify their acidophilic granules and bilobed nuclei!

Does anyone have a protocol that they've used sucessfully here?



#84065 Antibody staining

Posted by Piersgb on 19 August 2010 - 01:57 AM in Flow Cytometry

I think you can only really add a mixture of antibodies if you know that they will not cross-react and that you can also detect them separately and clearly. You'd also need a pretty good flow cytometer with several different lasers/detectors to then be able to differentiate between which antibody has bound and which is fluorescing in the mixture.

I think it would be best to start adding antibodies individually so that you can say for sure that any result you are getting is due to the one antibody that you have added. I'm assuming that you are also using isotype controls?

I agree with everyone else about the tyrpsin. To protect your membrane bound proteins it would be sensible not to use trypsin. You could always try this at a later date to see how using trypsin affects your results.



#84063 HBSS replacement for washing steps in antioxidant assay using flow cytometry

Posted by Piersgb on 19 August 2010 - 01:30 AM in Cell Biology

The change in buffer may have some effect on your flow results, the best way to find out is to compare the results of the same stainings with different buffers yourself with your samples. The difference between PBS and HBSS is shown below:


Google: "HBSS PBS recipe" :

HBSS
400 ml H2O

4.0 g NaCl (=13.7 ml 5 M NaCl)
0.20 g KCl
0.50 g Glucose
0.021 g Na2HPO4
0.027 g KH2PO4
0.096 g CaCl2
0.111 g MgCl2
0.176 g NaHCO3

qc 500 ml
pH 7.1
Filter sterilize

PBS
900 ml H2O
8 g NaCl or 27.4 ml 5M
0.2 g KCl
0.19 g KH2PO4 or 1.4 ml 1M
0.61 g Na2HPO4 or 4.3 ml 1M

qc 1 L
pH 7.4



#82618 hematopoietic cell lines

Posted by Piersgb on 06 August 2010 - 02:32 AM in Stem Cell

I'm looking at mouse haematopoietic stem cells. Does anyone else have experience with a good cell line?



#82615 Over high percentage of CD4+CD25+‏

Posted by Piersgb on 06 August 2010 - 02:13 AM in Flow Cytometry

What isotype are your antibodies to CD4 and CD25? If they are the same isotype as each other then you'd only need one isotype control? If not then you'd possibly need an isotype control for each of your antibodies.

Have you done any blocking/fixing steps in your protocol?



#82291 Washing Red Blood Cells

Posted by Piersgb on 03 August 2010 - 04:21 AM in Hematology

I guess it doesn't matter which Calcium chelator you use (?)

I read somewhere that you can draw the Calcium chelated blood into a syringe with the dextran added and leave for an amount of time to allow the RBCs to settle out. This would leave the plasma on top with the buffy coat inbetween the plasma and RBCs. Then you can attach a bent needle and carefully collect the layers from the syinge that have separated out.

More details were on:

www.malariasite.com/malaria/qbc.htm

I can't seem to get on this today though :S



#82289 Fixing cells for flow cytometry

Posted by Piersgb on 03 August 2010 - 03:33 AM in Flow Cytometry

What is the reason you want an alternative to PFA? My group tend to use 4% PFA in 1 x PBS for 10 minutes and then wash...

IHC world is usually quite good:

http://www.ihcworld....CC/fixation.htm




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