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Does anyone know how to attach cells to quartz cover glass or rather how to
coat the quartz so that the surface would become positively charged.

I am not sure, but quartz has a surface negative charge at >pH 2.9 ?

Vitaly
NCI-Frederick,
301-846-6575

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On
Behalf Of Mario Moronne
Sent: 20 сентября 2006 г. 15:35
To: [log in to unmask]
Subject: Re: A question about loosely attached cells

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Tian,

You have already received some of the classic tricks for improving 
attachment of cells to slides. I thought I might mention a couple 
more and qualify the use of polylysine. Regarding the latter, 
polylysine improves cell to glass binding by neutralizing the 
negative charges commonly observed on glass and the negative charge 
moieties on cell surfaces. Further this association helps form 
bridges between the cell surface and the glass.

Polylysine works best when it has a molecular weight of 300 kDa or 
more (~300 lysine groups). The reason is simply that the free energy 
of binding per lysine -NH2, whether to glass or protein carboxyl 
group, though very weak per monomer, increases exponentially when 
combined per additional -NH2, or more precisely, -NH3+. The 
individual subunits may bind poorly but when a couple of hundred 
subunits are combined, we suddenly go from say 1 KCal/mole to maybe 
200 KCal/mole affinity constant. A similar approach would be to 
pre-treat slides with an aminopropyl silane. In this case, the silane 
forms a covalent bond to the glass and an electrostatic one to the 
cells. Some companies make prepared slides, so you might buy some 
first to test to see if they will work. Just a word of caution, -NH2 
can become oxidized and you lose the electrostatic cling. I always 
make my own.

A couple of other approaches, include the application of Matrigel (an 
ECM preparation). This I have found to create problems in that the 
adhesion is fine but it can seriously affect the cell morphology and 
filamentous structures. My overall favorite except for very specific 
applications is pre-treatment of slides or coverslips with 
fibronectin (5 ug/ml), let them dry, apply medium and attempt to 
attach cells. With some cells you can just add the fibronectin to the 
media and wait half a day.

In general, this is a topic that has been discussed many many times 
on the list; I suggest you search the archives. Sometimes you just 
have to try lot of different approaches until you get what you want 
for your particular cells.

Let us know what works for you,
Mario

>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>I am using chamber slide for confocal studies. The cell line I use is a
>RAW 264.7 cell line, these cells are only semi-attached to the slide. To
>make matters worse, I use toxic stuff as the treatment, some attaching
>cells will become detached, and no matter how careful I do washing, fix,
>staining, a large number of cells will get lost and you can only find a
>bunch around the corner of the chamber slide. Does anyone know how to deal
>with these loosely attatch cells? Thanks a lot!


-- 
____________________________________________________________________________
____
Mario M. Moronne, Ph.D.
ph (510) 528-2400

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