Neutral red isn’t actually a good stain for single cell resolution (even in development, it’s pretty tricky - overstaining can impede development). But because it depends on pH changes in the vacuole, it’s good for distinguishing cell types in a slug, but not so great when trying to resolve single cells. 

You may have better luck with Nile blue sulfate. 



On Dec 18, 2013, at 11:43 PM, Jon Moore <[log in to unmask]> wrote:

> Dear George and colleagues,
> 
> I've got a lot of good recommendations, and sorting through them all will take time.
> Perhaps I've been violating the conventions of the listserv, but generally I've been 
> responding directly to each recommender.
> 
> Responding to a few of the recent ones:
> - We tried pre-incubating cells with neutral red, but they tended to lose it during 
> migration time, making them actually harder to see.  Perhaps, though, it would
> be worth trying to put it into the media at a low concentration.
> - One reasons we changed to dicty for this lab from a human cancer cell line
> was risk mitigation when dealing with ~140 mostly freshmen every year.
> Adding Serratia marcescens seems dubious.
> - We're use that slightly out of focus phenomenon.  That's hard to teach, though,
> they're simply having a hard time identifying them.
> -Spencer's idea of using florescence has a lot of of appeal, especially in terms 
> of the wow-cool factor.  I got a GFP containing strain from the stock center:
> Strain-DBS0236176 - [act15]:GFP:act15
> I was going to try it with a collaborator's NightSea system, but I found that I 
> needed more power than I thought NightSea would provide when I looked at this strain
> using the communal high powered florescent 'scopes.
> -I noticed the stock center doesn't have a histone-tagged GFP.  I use one for
> my research and it works like a charm.  If you're still reading, might you have one?
> -And, yes, David is spot on about our trouble.  We modeled this lab on one
> he uses in his cell bio course, but with a ton of tweaks for our equipment,
> our larger younger cliental, and our learning goals.
> 
> I've provided the relevant section of last year's course manual to some of
> you.  If you'd like that, I'll happily share directly.  It isn't obvious to me how to 
> do that through the listserv.
> 
> Peace and many thanks!
> 
> Jon
> 
> 
> 
> From: George McNamara <[log in to unmask]>
> Organization: George McNamara
> Reply-To: "[log in to unmask]" <[log in to unmask]>
> Date: Wednesday, December 18, 2013 5:58 AM
> To: Jon Moore <[log in to unmask]>
> Cc: "[log in to unmask]" <[log in to unmask]>
> Subject: Neutral red live cell stain ... Re: [DICTY] dicty microscopy for a teaching lab
> 
> Hi Jon,
> classic Dicty literature used Neutral red to stain live cells. A google search turned up many hits, one to S. Matsukuma, was "incubating in 0.005% neutral red solution for 10 minutes before spreading"
> 
> A much more risky approach is to feed the Dicty on Serratia mascarens (may have species name misspelled), but bad things could happen if the wrong strain and/or poor cell culture techniques were used.
> 
> tips for unstained cells:
> * Jeff Segall published many years ago on deliberately being out of focus to make the dicty amebae higher contrast. This affects image quality - not going to see pseudopod details - but contrast is critical. Contrasty out of focus cells are more useful than invisible cells. Paper was (probably):
> Quantification of motility and area changes of Dictyostelium discoideum amoebae in response to chemoattractants.
> Segall JE.
> J Muscle Res Cell Motil. 1988 Dec;9(6):481-90.
> PMID:  2850298
> 
> * consider doing image processing to bring out details. Could use the Fiji ImageJ distribution for this (free at http://fiji.sc/Downloads ). Acquire an empty field of view (of similar setup without cells). Background subtract (plus offset) and enhance contrast. 
> 
> 
> 
> If you ever decide to "go live" (web cam?) ...
> 
> If you get live imaging optimized:
> - check out my video "The Chase" http://works.bepress.com/gmcnamara/18/  (also available is  http://works.bepress.com/gmcnamara/4/ which is smaller but less informative) - it was of a neutrophil, but the Temporal Area Map (TAM) and histogram was developed for dicty. 
> 
> More information in my newsletter articles at
> http://mdc.custhelp.com/euf/assets/content/MetaMatters%20vol%202_iss3.pdf
> http://mdc.custhelp.com/euf/assets/content/MetaMatters%20vol%202_iss4.pdf
> http://mdc.custhelp.com/euf/assets/content/MetaMatters%20vol%202_iss5.pdf
> http://mdc.custhelp.com/euf/assets/content/MetaMatters%20vol%202_iss6.pdf
> 
> Tomasz Zal's lab discovered TAM on their own and published in:
> Body-barrier surveillance by epidermal γδ TCRs.
> Chodaczek G, Papanna V, Zal MA, Zal T.
> Nat Immunol. 2012 Feb 12;13(3):272-82. doi: 10.1038/ni.2240.
> PMID:  22327568  
> 
> Nikon objective lenses have threads that are close enough to C-mount format that you could attach the objective lens directly to a C-mount camera -- there might even be some "web cams" that have C-mount adapters. This would eliminate the microscope body. Could use another lens on the other side. 
> 
> You might also find inspiration in the Protasenko et al microscope at
> http://chemeducator.org/sbibs/s0010004/spapers/1040269mk.htm
> 
> Some labs and core facilities are retiring their old video and digital cameras, for example, Hamamatsu ORCA-ER CCDs. The Dicty community (DictyBase?) has or can start an educational exchange where retired 'research' equipment can be re-purposed for education. Micro-Manager / (Fiji) ImageJ supports a lot of hardware http://micro-manager.org/wiki/Device_Support
> You can also ask vendors if they have taken in equipment as trade-in offers.
> 
> Sincerely,
> 
> George
> 
> 
> On 12/17/2013 2:59 PM, Richard Sucgang PhD wrote:
>> Hello, Jon,
>> 
>> I am glad you are using Dicty as a teaching tool in Pomona. 
>> For your purposes, staining with crystal violet or Coomasie may be the most appropriate (with fixation, I think). 
>> 
>> 
>> http://dictybase.org/ListServ_archive/listserv_archive_staining.html
>> 
>> 
>> See the section from Dale Hereld. 
>> 
>> -Ricky
>> 
>> On Dec 17, 2013, at 11:45 AM, Jon Moore 
>> <[log in to unmask]>
>>  wrote:
>> 
>>   
>> 
>>> Last spring we started running a dicty motility teaching lab for an intro cell
>>> biology/ cell chemistry class.  We've been using a modification of the under-agar
>>> assay, and are happy with the lab with one major exception: it is very difficult
>>> for our students to see and and identify the cells with the limited microscopic
>>> power we have.  We're using dissecting microscopes with 50x total power, of which
>>> we have 19 research-grade. We have considered investing in better microscopes, 
>>> but there is little likelihood that we'll get 9 or more of these without outside funding.  
>>> We've tweaked the lighting about as much as possible.  Does anyone have any
>>> suggestions as to how we might better visualize them, including ideas like good 
>>> stains?  We're running this as an end-point assay, so if the stain kills them once
>>> they've migrated, that's Ok.
>>> 
>>> Thanks and peace,
>>> 
>>> Jon Moore
>>> assistant professor, Pomona College
>>> 909-621-8607
>>> 
>>> [log in to unmask]
>>> 
>>> 
>>> (Yes, I've met almost none of you.  No one at Pomona works on dicty;
>>> this is an outgrowth of a lab that started with a breast cancer cell line.)
>>>     
>>> 
>>   
> 
> 
> -- 
> 
> 
> 
> George McNamara, Ph.D.
> Single Cells Analyst
> L.J.N. Cooper Lab
> University of Texas M.D. Anderson Cancer Center
> Houston, TX 77054
> Tattletales 
> http://works.bepress.com/gmcnamara/26/
>  
>