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wjchxl's Content

There have been 9 items by wjchxl (Search limited from 22-November 18)


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#82421 recommendation wanted for microscope

Posted by wjchxl on 04 August 2010 - 08:38 AM in Tissue and Cell Culture

Hi,

Information is wanted for a microscope on which a digital camera may be installed for tissue culture study. Any recommendation on models would be really appreciated. The more details, the better.

Thanks!



#78351 question about qRT-PCR preparation

Posted by wjchxl on 06 July 2010 - 11:31 AM in PCR, RT-PCR and Real-Time PCR

I use ABI one step qRT-PCR system. I am hesitating between two ways of setting up the final reaction mixture.

1) prepare a tube of mixture for triplicate reaction for each sample. The final step is to add 20ul final reaction mixture into the 384-well plate;

2) prepare a master mixture for all reactions; add RNA samples (in triplicate) into the 384-well plate. The final step is to add the aliquot of master mixture into the wells which already have RNA samples in them.

I felt the second way is easier than the first way, especially when I have many RNA samples and quite a few genes of interest. But with RNA samples in the 384-well plate (= open to the air) all the time until addition of the master mixture, I worry about any possible contamination/degradation.

Which way do you guys use? I'd appreciate any suggestions on this issue! Thanks in advance!



#76987 cell aggregation?

Posted by wjchxl on 25 June 2010 - 06:34 AM in Tissue and Cell Culture

I have been working on a stable LNCaP cell line. Recently I noticed cells appear to aggregate on the plate.

Attached picture 1 is the cell line with normal look. picture 2 is the cell line that has the "aggregation look".

What does this kind of phenotype suggest?

Thanks!

Attached Thumbnails

  • 2.jpg
  • 1.jpg



#76982 cell adhesion in serum free condition

Posted by wjchxl on 25 June 2010 - 06:26 AM in Tissue and Cell Culture

By the way, it is LNCaP cells. Any suggestion?



#76307 stability of cell suspension

Posted by wjchxl on 21 June 2010 - 07:13 AM in Tissue and Cell Culture

Hi,

I need to prepare cell suspensions of seven cell lines and use them at the same time for some experiment. Preparation for each cell line takes at least 30min. So I am wondering where to keep the prepared cell suspensions when working on others. Should I keep them on ice, on bench at room temperature, or 37C/5% CO2 incubator? Can they be stable for several hours?

Thanks a lot!



#76294 cell adhesion in serum free condition

Posted by wjchxl on 21 June 2010 - 05:01 AM in Tissue and Cell Culture

Hi,

I need to do check proliferation of my cell lines in serum free condition. So I have two choices:

1) trypsinize cells and resuspend cells in 10%FBS medium, plate cells; change medium to serum free the next day;
2) after trypsinizing and resuspending in 10%FBS medium, centrifuge cells, aspirate 10%FBS medium; resuspend cells in serum free medium and plate cells at desired amount.

I would prefer choice 2) since washing and changing medium may cause loss of this specific cell line. But I don't know if cells can adhere to the plate when plated in serum free medium.

Any suggestions? Thanks!



#76293 effects of cell confluency

Posted by wjchxl on 21 June 2010 - 04:54 AM in Tissue and Cell Culture

When I read papers, I noticed that sometimes authors mentioned about the cell confluency. For example, they may isolated RNA for microarray experiments "when cells are about 60% confluency"; they may collect protein lysates (to check phosphorylation level) "when cells are about 80% confluency". Why cell confluency matters? Sometimes to have as much lysates as possible, I let cells grow to almost complete confluency, would that affect my results?

Thanks in advance!



#66668 questions about soft agar assay

Posted by wjchxl on 14 April 2010 - 10:45 AM in Tissue and Cell Culture

I have been trying to do soft agar assay, which is a pain to me, especially when I have several cell lines. I have a couple of questions. I hope experts here can help me out.

Question 1: I saw people use agarose on some protocols. Is that the same thing as agar?

Question 2: how many times I may reheat the agar without affecting the experimental result? When I have several cell lines, the remaining top agar always set after I was done with the first cell line. I am wondering if I may reheat it again and again.

Question 3: after adding the top agar with cells onto the bottom agar, should I put the plates at 4C to solidify or just 37C incubator?

Question 4: what do you guys use to visualize the colonies? I have seen crystal violet or MTT on different protocols. Any preference? what concentrations for each? Time of incubation?

Thanks in advance!!!



#50878 freezing during RNA isolation step

Posted by wjchxl on 11 December 2009 - 01:08 PM in Molecular Biology

Hi,

I am using RNeasy kits from Qiagen for RNA isolation. I am wondering if the samples could be frozen down after lysis step. If yes, stored at what temperature? What risks are associated for long-term storage?

Thanks!




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