Stem Cell Method Discussion http://www.protocol-online.org/forums/index.php Fri, 20 Nov 2009 13:34:51 -0800 360 mitomycin c going into solution http://www.protocol-online.org/forums/index.php?showtopic=11699
I am using mitomycin c to make my MEF feeder layers for iPS cells. I ordered a 2mg vial from Sigma and resuspended in 2ml of water. I vortexed the vial to make sure the powder went into suspension completely. I aliquoted them out at 100ul each. However, after freezing I noticed that some of the vials contained blue-gray suspensions while others looked like the mitomycin had fallen out of suspension (there were blue flecks in clear solution).

Has anyone else experienced this? I have read that suspending in PBS is also done. Can I still use the aliquots with the blue flecks and hope they dissolve in the culture media? I'm trying to avoid irradiating the cells so any advice for preparing the mitomycin c would be greatly appreciated. Thanks!!]]> Fri, 20 Nov 2009 09:45:40 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11699 hematopoietic cell lines http://www.protocol-online.org/forums/index.php?showtopic=11686
Anyone here has experience with hematopietic cell line expansion? I am about to buy one, but I do not know which is good..does any one has recommendation?

Past few months, i cultured cord blood hematopoietic stem cells. The problems: cells didnt expand much after 1-2 weeks upon expansion and couldnt be maintain in its undifferentiated stated. I used serum-free media and cytokine cocktails from stemcell technologies. So I was thinking, why not I try with the cell line.

Thanks in advanve for any responses..

Rgrds,
Boudou]]> Thu, 19 Nov 2009 21:23:22 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11686 iPS media http://www.protocol-online.org/forums/index.php?showtopic=11434
Is there a pH indicator for stem cell media? I am used to using phenol red in my culture medium but the recipe I am using for iPS cells does not include it. See below for 500ml total volume.

Final Concentration
DMEM/F12 5.85g
Knockout replacement serum 100ml 20%
L-glutamine 5ml 2mM
Nonessential amino acids 5ml 0.1 mM
2-mercaptoethanol 3.5ul 0.1 mM
bFGF, 0.5ug/ul 10ul 10ng/ml
Pen/Strep 2.5ml 50U/ml, 50ug/ml
Sodium bicarbonate 1.22g

Thanks!!
]]> Mon, 09 Nov 2009 15:42:24 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11434 Gelatin coated dishes http://www.protocol-online.org/forums/index.php?showtopic=11355
I am new to working with stem cells and have a concern about gelatin coating. I made 0.1% gelatin and added 1ml/well of a 6-well plate. I incubated the plate o/n at 37deg. I aspirated the excess gelatin but it appeared that I had removed the entire volume without leaving much of a "coating."

How much of a coat should there be? Should there be a layer of gel at the bottom of the well or does it always remain a liquid? Also, I've seen that gelatin incubation can be completed in 15min. What difference does the length of incubation time make?

Thanks for the help!!]]> Wed, 04 Nov 2009 12:53:58 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11355 Experience with supersomes or microsomes http://www.protocol-online.org/forums/index.php?showtopic=11319
Does anyone has experience in working with Microsomes and supersomes.
I have problems using them in cell culture.
Need advice in using them

Thank you
Regards
Swarna]]> Tue, 03 Nov 2009 07:26:20 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11319 Tranfection of HL 60 cells http://www.protocol-online.org/forums/index.php?showtopic=11318
I would like to know if there is any protocol available for transfection of HL 60 cells.
and would like to know how many days will it takes to make a stable cell line

Thank you
Regards
Swarna
]]> Tue, 03 Nov 2009 07:18:10 -0800 http://www.protocol-online.org/forums/index.php?showtopic=11318 Mouse Anti-Brachyury antibody http://www.protocol-online.org/forums/index.php?showtopic=11258 I am looking for a good mouse Anti-Brachyury antibody for IHC. Do you have any suggestions? Fri, 30 Oct 2009 10:18:12 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11258 Distributer of GFP+ murine MSC http://www.protocol-online.org/forums/index.php?showtopic=11076
I am looking for some companies or institutions which is selling GFP+ mouse BM mesenchymal stem cell.
I used to buy them, but after I change my job to different university, I lost the old contact list, so I need your help.

I know that most of people will isolate by themselves, but currently I prefer to just have cell line.

So, if you know any place, please let me know.


Thanks]]> Fri, 23 Oct 2009 13:46:06 -0700 http://www.protocol-online.org/forums/index.php?showtopic=11076 Fetal liver or bone marrow? http://www.protocol-online.org/forums/index.php?showtopic=10915 My gene of interest is found frequently mutated in MDS and CMML patients. So I am about to study its role (self-renewal and malignant transformation) in HSC by KD and OE of mutants. But I am completely a green hand in this field. First I am wondering how I should isolate the HSCs, from Embryonic fetal liver or just adult mouse bone marrow? Thanks in advance. Fri, 16 Oct 2009 13:46:56 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10915 Reprogramming http://www.protocol-online.org/forums/index.php?showtopic=10843
In our lab we have quite a few Promoter driven Cre recombinase mouse lines that we often cross with RosaYFP. I was thinking of reprogramming some cells from these mice using a group of transcription factors and use the YFP as a readout. Typically in the literature a antibiotic resistance marker is used or a direct knockin cell line (Nanog-GFP). Would it be possible for a RosaYFP reporter to work?

Archdupass]]> Tue, 13 Oct 2009 19:02:42 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10843 Hematopoietic lineage depletion kit http://www.protocol-online.org/forums/index.php?showtopic=10762
I have a question concerning hematopoietic stem/progenitor cells enrichment kits. Has anybody tried to do lineage depletion in spleen or bone marrow to enrich Hematopoietic stem/progenitor cells. If yes, which kits have you used? I know there are many kits out there but I will be grateful to hear from someone who had experience.

Many thanks in advance,
berricom]]> Thu, 08 Oct 2009 20:16:34 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10762 time constant in electroporation http://www.protocol-online.org/forums/index.php?showtopic=10714 I would like to know how important the time constant in electroporation of ES cells.

I'm using biorad gene pulser -II for electroporation. I use 240 volts and 500micro farady for electroporation. Every I get a time constant 8.2-8.8. Is this normal?

thanks

best
mm]]> Wed, 07 Oct 2009 07:06:20 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10714 Haematopoietic stem cells http://www.protocol-online.org/forums/index.php?showtopic=10710 how to store haematopoietic stem cells for long term use????]]> Wed, 07 Oct 2009 05:45:11 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10710 Embryoid bodies http://www.protocol-online.org/forums/index.php?showtopic=10408 I am trying to differentiate mouse ES cells (E14-TG2A or D3 line). Any lineage would be OK. The Wobus et al 1997 protocol for cardiomyocytes did not give me more than one hollow sphere and no beating cells after: 2 days hanging drop, 8 days in bacteriological plate -LIF, then 9 days on gelatin -LIF +10-8M all trans retinoic acid. Any ideas? Should I have gotten rid of the MEFs for several passages before the hanging drop expt? Or avoid trypsin (they got very confluent in the center of the 24 well plates towards the end)? Mon, 21 Sep 2009 13:12:41 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10408 problem with MEF on 96 well plate http://www.protocol-online.org/forums/index.php?showtopic=10360
i'm using gamma-irradiated MEF as feeder layer for culturing ES cells. The MEF looks normal (with monolayer) on 6 well and 10cm plate. However, we see some morphological structures similar to small ES colonies when we place them on a 96-well plate (from falcon and greiner).


are there any other people have similar issues with MEF grown on 96 well plate or any suggestions how to deal with this.

thanks
m]]> Fri, 18 Sep 2009 01:02:44 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10360 self-renewal Vs proliferation http://www.protocol-online.org/forums/index.php?showtopic=10159
I havenīt posted for some time. I guess that is why my nick was cancelled. Anyways I created a new one. Lol

My question is regarding stem cells:
What is the difference between 'self-renewal' and 'proliferation'?
Is it possible to distinguish experimentaly self-renewal and proliferation? The assays that are performed in the literature (spheres cultures, serial passages of cells in animals) prove that cells have proliferation ability or self-renewal?


Thanks guys,
BioPe.
rolleyes.gif

]]> Mon, 07 Sep 2009 08:11:22 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10159 Collagenase http://www.protocol-online.org/forums/index.php?showtopic=10098 I'm trying to make a protocol about a preparation of collagenase solution, intended for adipose tissue digestion.
Iīve found that common used concentration is 0,1%.
Should I make a 0,1% solution (0,1mg/mL), indepedently of activity units?
What should I follow? Concentration or enzymatic activity?
Relatively to enzymatic activity, anyone knows what is PZ units?
Does anyone have a protocol about this?
Thanks a lot for your help.]]> Thu, 03 Sep 2009 08:53:40 -0700 http://www.protocol-online.org/forums/index.php?showtopic=10098 Adipose-derived stem cell http://www.protocol-online.org/forums/index.php?showtopic=9995
1. In some journals, they mentioned that adipose-derived stem cells would be favour for low % FBS (5-10%) instead of using high % of FBS (above 10%), is it right?

2. How can I collect more stem cell secretion?

Many thanks]]> Sat, 29 Aug 2009 06:44:05 -0700 http://www.protocol-online.org/forums/index.php?showtopic=9995 stem cells and EB (Embryoid bodies) http://www.protocol-online.org/forums/index.php?showtopic=9899
Another things, is the b-fgf very crucial for the stem cell differentiation respressing? I seem to have like add abit less than expected


PLS HELP!!!!]]> Tue, 25 Aug 2009 00:38:13 -0700 http://www.protocol-online.org/forums/index.php?showtopic=9899 FBS for stem cells http://www.protocol-online.org/forums/index.php?showtopic=9610 Any help, please?]]> Sat, 08 Aug 2009 19:52:52 -0700 http://www.protocol-online.org/forums/index.php?showtopic=9610