http://www.protocol-online.org/forums Fri, 10 Feb 2012 09:13:01 +0000 360 http://www.protocol-online.org/forums/topic/24368-ra-differentiation-of-sh-sy5y/
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]]> Fri, 10 Feb 2012 09:13:01 +0000 http://www.protocol-online.org/forums/topic/24368-ra-differentiation-of-sh-sy5y/ http://www.protocol-online.org/forums/topic/24348-to-treat-cells-with-inhibitor/
I just want to ask about some calculations about varying concentrations of drug treatments to my cells. For example, i have a 1uM stock of the drug and I need to treat the cells (3x10^3) in 3 cm dish with 100 nM in 2 ml of medium.

Does that mean i just simply dilute my drug to 100 nM from my 1uM stock and add it directly to my cells? or do i need to compute and consider the 2 ml of medium in the dish?

if you can share with me links or books to do math and computations of concentrations, i would be happy to know. thank yoou...]]> Thu, 09 Feb 2012 02:53:44 +0000 http://www.protocol-online.org/forums/topic/24348-to-treat-cells-with-inhibitor/ http://www.protocol-online.org/forums/topic/24324-culturing-cells-on-a-slide/ ,
In the beginning, I am very grateful for any one who will read my topic.

Secondly, I want to buy some culture wares for visualization of my cells under fluorescence or phase contrast microscope.
what I found due to reading some books and online:

*There are special type of culture slide such as
http://www.bdbiosciences.com/ecat/Searchresults.do?pgNum=1&pgSize=&sort=SortOrderDef&check=mainsearchcheck&key=cell+culture+slide&x=15&y=4&mterms=true

*It is untreated.
I should treat the surface with Poly L lysine or collagenase as I am working with adherent cells
but I cant know the difference between these reagent and which on is the best.

* After culturing, Does the step of fixation is necessary, or adhesion to surface of my slide is enough.
* Shall I use the ordinary glass cover on my slide or use these precoated glass covers or precoat them by myself.
http://www.bdbiosciences.com/ptProduct.jsp?prodId=364748&key=cell+culture+slide¶m=search&mterms=true&from=dTable

finally, does these steps differe if I will use a laser scanning confocal microscope?? has any one has experience with it and ready to share??

At the end I am deeply indebted for those who will share their thoughts, experience with me.

Best Regards]]> Wed, 08 Feb 2012 04:03:22 +0000 http://www.protocol-online.org/forums/topic/24324-culturing-cells-on-a-slide/ http://www.protocol-online.org/forums/topic/24322-cancer-biology-and-growth-factor-mechanisms/ Tue, 07 Feb 2012 22:05:13 +0000 http://www.protocol-online.org/forums/topic/24322-cancer-biology-and-growth-factor-mechanisms/ http://www.protocol-online.org/forums/topic/24239-low-signal-after-fixation-and-permeabilization/
I am transfecting my cells with alexa488 siRNA lipoplexes. Once I fix the cells (I tried 2.5 to 4% paraformaldehyde with 15-20 min incubation), the signal reduces with time. After actin phalloidin cell staining, that requires permeabilizing the cells, I see no green signal in the microscope (epi and confocal).
Actually I want to do colocalization studies of taken up siRNA with actin. How do I make this work? I know I cant use phalloidin for live cells. IS there any other way to stain actin in live cells except for using the method of transient transfection for actin?

Please help.

Mamcy40]]> Wed, 01 Feb 2012 05:07:49 +0000 http://www.protocol-online.org/forums/topic/24239-low-signal-after-fixation-and-permeabilization/ http://www.protocol-online.org/forums/topic/24209-problem-with-trypsinising-my-293ts/ Plz help.
Thank you so much]]> Mon, 30 Jan 2012 13:37:29 +0000 http://www.protocol-online.org/forums/topic/24209-problem-with-trypsinising-my-293ts/ http://www.protocol-online.org/forums/topic/24150-cytospin/
Thanks!]]> Wed, 25 Jan 2012 11:18:38 +0000 http://www.protocol-online.org/forums/topic/24150-cytospin/ http://www.protocol-online.org/forums/topic/24096-help-fitc-bsa/
I read that FITC (without BSA conjugation) decomposes in water (ref : sigma ), .?]]> Fri, 20 Jan 2012 09:03:52 +0000 http://www.protocol-online.org/forums/topic/24096-help-fitc-bsa/ http://www.protocol-online.org/forums/topic/24076-to-make-stable-cell-line-viral-system/
I wonder both advantage and disadvantage of sereral viral system such as lentiviral, retroviral and adenociral sysem.

]]> Thu, 19 Jan 2012 02:34:15 +0000 http://www.protocol-online.org/forums/topic/24076-to-make-stable-cell-line-viral-system/ http://www.protocol-online.org/forums/topic/24075-dose-response-troubles/
I have been performing dose response curves for certain chemotherapeutic that takes 3days to work on the cells. Earlier on a few months ago I was sucessful at obtaining nice curves with fairly low IC50 values. In the recent experiments I am not able to replicate my earlier results. My overall strategy is as follows, I have a stock solution of drug solubilized with DMSO at a stock concentration of 50mM. For cell culture, since DMSO is toxi, I vortex the solution and take 20uL of the stock and bring to volume with a 10mL volumetric flask using growth media which effectively is a X500 dilution. So the concentration would then be 100uM as the starting stock, I then do a series of 2X dilutions to get 50uM, 25uM, 12.5uM, 6.25uM, 3.125uM.

One thing which I have recently thought about which I am unsure of is the filtration step. Usually after the dilution into cell culture media I filter with a hydrophilic 0.2uM PVDF millipore filter using syringe into falcon tube. I am wondering if this is somehow trapping my drug and making that more dilute.

Earlier on I was obtaining IC50 values of ~5uM, recently that has shifted to around 50uM and something is really wrong with the results, as even the 100uM concentration has only around .50 response.

I am plating about 5,000 cells per well as 100uL in quadruplicate. Following day adding the drug solution, with incubation for 3 days, whereby I add MTT and check the results.

Any idea as to what is going on?

I already made a fresh solution and performed the experiment with similar results..... Could the filtration be an issue? Maybe the cells have been passed too long and have morphed into different state?


Any advice, tips or tricks ?

Thanks]]> Thu, 19 Jan 2012 02:27:09 +0000 http://www.protocol-online.org/forums/topic/24075-dose-response-troubles/ http://www.protocol-online.org/forums/topic/24021-any-methods-to-evaluate-the-attaching-ability-of-suspension-cells/
Since this cell line originated from T cell leukemia so normally it doesnt have attaching ability. And also as a T cell line, migration is a basic ability of it. So the first thought coming to my mind is that knockdown of this gene might reduced the migration of this cell line and enhanced the attaching ability of it to certain kinds of matrix like ECM, although I know there is no such layer and just a normal culture plate from Nunc. Indeed, this gene has been reported to be linked to tumor invasion and in normal cells it also has a role in cell morphological development. So, is my guess reasonable?

What I really want to ask is how can I prove my hypothesis? I have thought of staining and counting the attached cells and showing the difference as a data. But I feel that would not be a convincing data, or maybe not a real data that you can put in the paper at all. So, are there other methods for testing the attachment of cells? By the way, since we already have the plan checking its migration/invasion ability by boyden chamber, so I just want to know a method to address one point, how to quantify this attachment difference?

Sorry it is a little bit too long and thank you for reading it. If you have any idea, please tell me. Thank you very much.]]> Sun, 15 Jan 2012 10:11:15 +0000 http://www.protocol-online.org/forums/topic/24021-any-methods-to-evaluate-the-attaching-ability-of-suspension-cells/ http://www.protocol-online.org/forums/topic/23973-questions-on-life-science-homework-looking-for-explanations/ A)chloroplasts
B)rough endoplasmic reticulum
C)lysosomes


the word fluid in the term fluid mosiac model refers to the fact that
A)membranes are selectively permeable
B)the proteins within a membrane freely move around
C)the membrane assembles and disassembles as necessary for cell function


the proposed role of capsaicin in anesthesia during surgery is to
A)open channel proteins found only in pain-sensing neurons allowing an anesthetic to enter those cells alone
B)increase the rate at which calcium ions enter a nerve cell
C)block the transmission of electrical signals between nerve cells and muscle cells

Thank you very much. I can't quite grasp these concepts and the homework spans a few lessons' material I seem to be missing]]> Tue, 10 Jan 2012 22:44:27 +0000 http://www.protocol-online.org/forums/topic/23973-questions-on-life-science-homework-looking-for-explanations/ http://www.protocol-online.org/forums/topic/23964-detection-of-ros-by-dcfda/ Although application of NAC, a well known antioxidant significantly counteract the effect of my chemical, i always suffer one problem that the chemical shows no obvious influence on ROS level when using DCFDA or DHE to detect. It is certain that DCFDA works since the basic level of ROS was detectable. Is it possible that my chemical induces extraordinary ROS than the normal one?

The detail of experiment procedure is as follows:

Cells cultured in RPMI supplemented with 10% FBS and 1% P/S were treated with my chemical for 24 hours. Cells were washed with PBS, trpsinized, and neutralized with FBS. After washing with PBS again, cells were incubated in PBS containing 10 uM DCFDA for 30 min. After washing again, cells were analyzed using flow cytometry.

I had also tried to stain cells without trypsinization; however, the result remain similar with no significant difference.

Any suggestion?

Thank You]]> Tue, 10 Jan 2012 09:10:57 +0000 http://www.protocol-online.org/forums/topic/23964-detection-of-ros-by-dcfda/ http://www.protocol-online.org/forums/topic/23957-transfection-problem-after-transformation/ I am trying to amplify a plasmid (P3XFLAG-CMV-14 plus non toxic insert) through backterial transformation. The plasmid that I am using works great for transfection in HEK293T cells, however, after transformation and DNA extraction, the HEK cells don't express the protein at all (or at very very low levels).

I have sequenced my insert and the CMV promoter and they are fully intact.

I have linearized my construct and it migrates at the same rate as the transfectable one.

I don't believe endotoxin is a problem, since I've tried using DNA extraction kits with and without endotoxin removal buffer. Plus http://www.ncbi.nlm....pubmed/10997275

I have tried different chemical competent cells (DH5-alpha, JMH109 & XL10 Gold) without success.

I appreciate any inputs!! ]]> Mon, 09 Jan 2012 14:32:36 +0000 http://www.protocol-online.org/forums/topic/23957-transfection-problem-after-transformation/ http://www.protocol-online.org/forums/topic/23941-help-with-bt549-morphology-maintenance-possible-contamination/
I'm currently an undergrad student working on BT549 & MB231 to study signalling pathways. However, I've encountered much difficulty in maintaining sufficient and healthy BT549 for my work.

Growth Characteristic
Does anyone have any idea what the doubling time of BT549 is? My senior told me it was similar to MB231, which is 36H. But ATCC does not mention this, so I was hoping if someone could verify this for me. One of the growth characteristics I noticed with my BT549 is that they require a very high seeding density in order for them to grow well (confluent T75 = 2xT75 or less), have anyone experienced this problem as well? The BT549 I'm growing also seem to slow down a lot in their growth towards the later passages, and I'm not sure if this is due to genetic instability of the cell line, maintenance issues or contamination, could someone help verify on this?

Morphology
I was doing a search online to find images of what BT549 should look like for my project that requires me to work with this cell line. Apparently ATCC does not have an image to show what it's morphology is like, but only describes that it should look like epithelial cells.

Maintenance
Although ATCC recommends the supplementation of insulin for the maintenance of BT549, the lab I'm working in does not use insulin to maintain this cell line. The growth characteristic of the BT549 that we maintain seem to look like elongated versions of MB-231, with a very thin and needle-like body, not sure if it is suppose to be this way. I'll try to get a picture my the BT549 and upload it soon.

Also, in the course of maintaining BT549, i realized that a lot of black dots seem to appear in the back ground. Not sure if this is a characteristic of this cell line or contamination. But it could well be cell debris moving in the background through Brownian motion. I'll try to get an image of this up soon as well.

Much help is appreciated. Thanks.

Regards,
Unknownko]]> Sat, 07 Jan 2012 14:50:17 +0000 http://www.protocol-online.org/forums/topic/23941-help-with-bt549-morphology-maintenance-possible-contamination/ http://www.protocol-online.org/forums/topic/23924-macrophage-immortalization/
can anyone give me a good protocol for macrophage immortalization,derived from mice bone marrow?Which virus to use is the best?

thank you,

dusko]]> Fri, 06 Jan 2012 06:51:02 +0000 http://www.protocol-online.org/forums/topic/23924-macrophage-immortalization/ http://www.protocol-online.org/forums/topic/23908-where-can-i-find-more-info-on-how-certain-ionic-solutions-can-affect-cell-biology/ In many protocols involving some form of treatment on cells, for example immunostaining protocols, I run into many 'recommended' solutions that the protocols suggest me to use on the cells for certain steps such as a pre-extraction step for immunostaining MDCK's tight junctions.

Buffer:
* Pre-extract with 0.2% Triton X-100 in 100mM KCL, 3mM MgCl2, 1 mM CaCl2, 200mM sucrose, and 10mM Hepes (pH 7.1) for 2 min on ice, making sure that buffer is gently applied to cells.

I know this step is definitely crucial but for curiousity's sakes as well as for me to simply learn more about how certain solutions with certain ions and their concentrations affect - structure + physiology, are there any certain websites or books where I can learn more about how these solutions affect cells?

I am very weak in cell biology, and any recommendations that may help me learn more about cells is greatly appreciated.

Thanks for the time!]]> Wed, 04 Jan 2012 18:46:45 +0000 http://www.protocol-online.org/forums/topic/23908-where-can-i-find-more-info-on-how-certain-ionic-solutions-can-affect-cell-biology/ http://www.protocol-online.org/forums/topic/23847-help-with-some-problems-with-culturing-epithelial-cells/
bobby has been culturing epithelial cells for experiments that he is performing in the laboratory. He has subcultured the cells many times, and has always fed the cells the proper medium. He recently ran an experiment and found that his cells are not growing the way that he previously observed. What are some reasons why these cells may not be acting as they did previously?

Since this was a free online lecture, the answer was not provided and since i am having the same issue in my lab, I am wondering what is the cause for this phenomenon and if you guys may know the answer. Also, if possible, where would I be able to read more about this?

Thanks.]]> Mon, 26 Dec 2011 01:30:18 +0000 http://www.protocol-online.org/forums/topic/23847-help-with-some-problems-with-culturing-epithelial-cells/ http://www.protocol-online.org/forums/topic/23834-nuclear-extract-problems/
May be someone could spare a bit of time to share experience on nuclear extract preparation. I tried to get it from primary leukocytes according to a published protocol that in principle involves 2 buffers. A brief incubation with a hypotonic buffer and NP40 to lyse the cells and then a longer lysis of the nuclear pellet. In the end however I got very little protein. The Bradford assay estimated only about 2ug per ml from 5 million cells. I think that I lost proteins in the course of the extraction. The protocol calls for resuspension of the nuclear pellet after the first lysis. I found it very difficult to do as the pellet was extremely sticky. I pipetted it up and down for 15 min, incubated on ice 20 min and then continued for to pipette for another 10 min, but there were still insoluble clumps present. Your advice is most appreciated! Any links to good protocols to get nuclear extracts from limited quantity of cells for emsa? Thank you! Merry Christmas!]]> Thu, 22 Dec 2011 16:54:12 +0000 http://www.protocol-online.org/forums/topic/23834-nuclear-extract-problems/ http://www.protocol-online.org/forums/topic/23804-lentiviral-packaging-systems/
I am new to virus work and packaging systems. I thank you if you could suggest me some useful links/pdf/ebooks to gain the background.

For example, I want to know which components is necessary for virus production and which vectors are compatible to which.

Thanks]]> Sun, 18 Dec 2011 20:32:45 +0000 http://www.protocol-online.org/forums/topic/23804-lentiviral-packaging-systems/