James -
Have you verified that this protocol is
sufficient to really equilibrate the mineral oil,
i.e. that the composition of medium is no longer
affect by contact with the equilibrated mineral
oil? Or are you just going by cell
survival/growth, which is certainly a good
indicator but doesn't necessarily mean that the
cells are affected in a non-lethal way?
Also, still need to consider the effects of the
mineral oil if you want to add compounds during
the experiment. BTW, I don't mean to question
that a mineral oil overlay is suitable for many
applications, but as always, it's good to be aware of possible side-effects.
Beat
At 23:17 26-02-2009, you wrote:
>Here is the protocol in use at The Jackson
>Laboratory for producing mineral oil for use in
>in vitro fertilizations and embryo culture:
>
>
>1. In a sterile bottle add 1000mL of oil and 100mL tissue culture medium.
>2. Stir oil/medium for two days at room temperature.
>3. Let oil/medium settle for one day.
>4. Decant medium the next day.
>5. Filter the oil using a 0.8ĂL filter
>(Fisher #09-740-30), aliquot into 250 ml units.
>
>The oil is stable for two months at room temp.
>
>Use it to layer over cells in medium, to prevent
>evaporation. It will exchange gases, allowing
>CO2 to maintain the proper pH (we have verified
>this using a pH indicator, and moving dishes in and out of CO2 incubators.
>
>
>
>James Denegre, Ph.D.
>Associate Research Scientist
>Manager, Light Microscopy
>The Jackson Laboratory
>Bar Harbor, ME 04609
>207.288.6321
>
>-----Original Message-----
>From: Confocal Microscopy List
>[mailto:[log in to unmask]] On Behalf Of Armstrong, Brian
>Sent: Thursday, February 26, 2009 11:46 AM
>To: [log in to unmask]
>Subject: Re: CO2, humidity, temp sensors and mu\ineral oil
>
>Hi James, can you elaborate on your technique " equilibrate the mineral
>oil with the medium"?
>Thanks,
>
>Brian D Armstrong PhD
>Light Microscopy Core Manager
>Beckman Research Institute
>City of Hope
>Dept of Neuroscience
>1450 E Duarte Rd
>Duarte, CA 91010
>626-256-4673 x62872
>http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imag
>ing/Pages/default.aspx
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[log in to unmask]]
>On Behalf Of James Denegre
>Sent: Thursday, February 26, 2009 8:10 AM
>To: [log in to unmask]
>Subject: Re: CO2, humidity, temp sensors and mu\ineral oil
>
>In our lab we equilibrate the mineral oil with the medium to prevent
>uptake of components, and have successfully imaged under mineral oil for
>five continuous days.
>
>James Denegre, Ph.D.
>Associate Research Scientist
>Manager, Light Microscopy
>The Jackson Laboratory
>Bar Harbor, ME 04609
>207.288.6321
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[log in to unmask]]
>On Behalf Of Beat Ludin
>Sent: Wednesday, February 25, 2009 5:05 AM
>To: [log in to unmask]
>Subject: Re: CO2, humidity, temp sensors
>
>Over time, the mineral oil overlay will basically void the medium of
>all lipophilic components (vitamin E, for instance). This may or may
>not be a problem, but you need to be aware of it and do the proper
>controls.
>
>If you want to check for evaporation, just weigh your sample before
>and after the imaging.
>
>Beat
>
>At 23:23 12-02-2009, you wrote:
> >My Guess is still evaporation of media is killing your
> >cells.. Overlay the cell media in each compartment with mineral
> >oil. It stops all evaporation but allows CO2 gas exchange to occur
> >to maintain pH.. I do this with all my chamber systems and it works
> >great. No need to humidify flowing air or gas.
> >
> >
> >Joe Goodhouse
> >Confocal Core Lab Manager
> >Dept. of Molecular Biology
> >Princeton University
> >609-258-5432
> >
> >Visit us at
> ><http://www.molbio/>http://www.molbio1.princeton.edu/facility/confocal/
> >
> >
> >
> >----------
> >From: Confocal Microscopy List
> >[mailto:[log in to unmask]] On Behalf Of Paul Herzmark
> >Sent: Thursday, February 12, 2009 4:36 PM
> >To: [log in to unmask]
> >Subject: Re: CO2, humidity, temp sensors
> >
> >
> >I knew I would not get away with out details!
> >
> >I have an Invivo (company) plexiglass incubator box around my
> >inverted microscope. 5% CO2 is piped inside to a small space (about
> >50 ml) around the multiwell-Labtech (company)-coverslip. The small
> >space is created by a glass plate with a frame around it that goes
> >over the coverslip.The CO2 passes into the small space via special
> >tubing.The tubing goes under incubator-warmed water where it is
> >supposed to pick up water vapor without bubbling.
> >
> >1. The whole incubator, with most of the microscope is warmed to 37
>degrees
> >2. Only the small space surrounding the cells is humidified and has
>CO2.
> >
> >
> >
> >
> >On Thu, Feb 12, 2009 at 11:30 AM, Goodhouse, Joseph G.
> ><<mailto:[log in to unmask]>[log in to unmask]> wrote:
> >Paul,
> > Could you describe in detail your heating system. Do you
> > overlay your cells with mineral oil? This prevents evaporation
> > which will alter your salt concentration.
> >
> > I use a Recordable Temp sensor from Fisher Scientific. Cat No
> > 15-007-26. Inexpensive and works well.
> >
> >
> >Joe Goodhouse
> >Confocal Core Lab Manager
> >Dept. of Molecular Biology
> >Princeton University
> >609-258-5432
> >
> >Visit us at
> ><http://www.molbio/>http://www.molbio1.<http://princeton.edu/facility/c
>onfocal/>princeton.edu/facility/confocal/
> >
> >
> >
> >
> >----------
> >From: Confocal Microscopy List
> >[mailto:[log in to unmask]] On Behalf Of Paul Herzmark
> >Sent: Thursday, February 12, 2009 2:24 PM
> >To:
><mailto:[log in to unmask]>[log in to unmask]
>U
> >Subject: CO2, humidity, temp sensors
> >
> >I have a cell dying problem in my microscope when I take pictures of
> >them overnight or longer. I suspect the humidity is failing and I
> >want to monitor that, CO2 and temperature.(It is not photodamage.
> >Cells die in areas away from the objective.)
> >
> >Can anyone suggest a supplier of recording sensors that I can get
> >next to the well slides on my stage?
> >
> >Thank you!
> >
> >Paul Herzmark
> >Specialist
> ><mailto:[log in to unmask]>[log in to unmask]
> >
> >Department of Molecular and Cell Biology
> >479 Life Science Addition
> >University of California, Berkeley
> >Berkeley, CA 94720-3200
> >(510) 643-9603
> >(510) 643-9500 fax
> >
>
>
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