 |
 |
 |
 |
----My original question:
>what is your preferred technique for keeping cells stuck to glass slides,
>post sorting, so as to permit subsequent fixing and morphological analysis
>by cytochemistry? Any technique that works from sorted droplet DIRECTLY to
>slide (that gets rid of saline), for those situations where there are too
>few sorted cells to reanalyze/ make cytosmear with?
> Does CELL-TAk , SUPER FROST , or some other sticky stuff work?
>thanks.
=============================================================
response #1
When sorting onto slides we use very dilute saline (10 ml of Isoton in 2
litres of distilled water). We then dry at 50 degrees. We do not coat
slides.
Cells adhere well. Droplet deflection is just as good.
Good luck.
_______________________________________________
Graham Vesey
Australian Environmental Flow Cytometry Group
School of Biological Sciences, .-.--:_:\
Macquarie University, _/ \
Sydney, : AEFCG |
Australia NSW 2109. \_ /
Tel- 612 850 8150 '-''''\__/
Fax- 612 850 8174 V
_______________________________________________
================================================================
response #2
Lan Bo Chen, around the corner from you at the Farber has some
magical slides that he gets from Germany. The slides are treated by
a proprietary method. He claims to retain >90%
of the cells through rigorous processing.
Albert D Donnenberg, PhD
University of Pittsburgh
Albert D. Donnenberg, PhD
Associate Professor of Medicine
====================================================================
response #3
Centrifugal Cytology works. I haven built special buckets. The slides
should be clean and then coated with polylysine or the best binding will be
with Meyer's Albumin fixative. The trick is to fix the cells while the
centrifugal field is applied. Glyoxal is a good fixative.
My technique, Centrifugal Cytology, is described in the papers listed below.
I still have reprints of many of the papers, which upon request, I will send
by conventional, snail, mail.
Eileen King's group, Livermore, and Los Alamos sorted directly into the
buckets. While I was at Coulter, I designed and built buckets which fir
into the EPICS.
1. R. C. Leif, L. M. Vallarino, and P. M. Harlow; Production, Fixation, and
Staining of Cells on Slides for Maximum Photometric Sensitivity".
Proceedings of Biochemical Diagnostic Instrumentation, Progress in
Biomedical Optics. Ed. R. F. Bonner, G. E. Cohn, T. M. Laue, and A. V.
Priezzhev. SPIE Proceedings Series 2136, pp. 255-262 (1994).
2. R. C. Leif, K. L. Chew, E. B. King, M. L. Cayer, and R. Cowden; "The
Potential Of Centrifugal Cytology Dispersions For Automated Cytology". The
Compendium on the Computerized Cytology and Histology, Chicago (1994).
3. D. Bobbitt, M. Silverman, A. Ng, M. Bondhus, C. Railey, D. Hindman, R.
C. Leif; "Centrifugal Cytology of Urine". Urology, 28 pp. 432-433 (1986).
4. R. D. Stulting, R. C. Leif, J. Clarkson, D. Bobbitt; "Centrifugal
Cytology of Ocular Fluids". Arch. Ophthalmol. 100, pp. 822-825 (1982).
5. H. G. Haines, J. P. McCoy, D. E. Hofheinz, A. B. P. Ng, S. P. B.
Nordqvist and R. C. Leif; "Cervical Carcinoma Antigen in the Diagnosis of
Human Squamous Cell Carcinoma of the Cervix". JNCI 66 pp. 465-474 (1981).
6. R. C. Leif, D. Bobbitt, C. Railey, V. Guarino, R. DerHagopian, A. Ng.
and M. Silverman; "Centrifugal Cytology of Breast Aspirate Cells". Acta.
Cytologica 24 pp.255-261 (1980).
7. R. C. Leif, M. Silverman, D. Bobbitt, A. Ng; "Centrifugal Cytology: A
New Technique for Cytodiagnosis". Laboratory Management 17, September, pp.
38-41 (1979).
8. R. C. Leif; "Buoyant Density Separation with Linear Gradients of Bovine
Serum Albumin and Analysis by Centrifugal Cytology and Flow Techniques". In
Methods of Cell Separation. Vol. 2., Edited by N. Catsinpoolas, New York,
Plenum, pp. 181-270 (1979).
9. R. C. Leif, D. J. Ingram, D. Bobbitt, R. Gaddis, S. Nordqvist and A. B.
P. Ng; "Centrifugal Cytology, Dissociation and Staining of Gynecological
Cells". In The Automation of Cancer Cytology and Cell Image Analysis, Edited
by H. J. Pressman and G. L. Wied, Tokyo, pp. 53-62 (1979).
10. R. C. Leif, S. Nordqvist, C. Clay, M. Cayer, D. J. Ingram, B. F.
Cameron, D. Bobbitt, R. Gaddis, S. B. Leif and A. Cabanas; "A Procedure for
Dissociating Ayre Scrape Samples". J. Histochem. Cytochem. 25, pp. 525-537
(1977).
11. R. C. Leif, D. J. Ingram, C. Clay, D. Bobbitt, R. Gaddis, S. B. Leif
and S. Nordqvist; "Optimization of the Binding of Dissociated Exfoliated
Cervico-Vaginal Cells to Glass Microscope Slides". J. Histochem. Cytochem.
25, pp. 538-543 (1977).
12. J. T. Thornthwaite, M. L. Cayer, B. F. Cameron, S. B. Leif and R. C.
Leif; "A Technique for Combined Light and Scanning Electron Microscopy of
Cells". Scanning Electron Microscopy, Vol. V, Edited by O. Johari and I.
Corwin, IIT Research Institute, Chicago, pp. 127-130 (1976).
13. J. T. Thornthwaite and R. C. Leif; "A Permanent Cell Viability Assay
Using Alcian Blue". Stain Technology 53, pp. 199-204 (1975).
14. L. A. Dunlap, R. L. Warters and R. C. Leif; "Centrifugal Cytology III:
The Utilization of Centrifugal Cytology for the Preparing Cells for Critical
Point Drying". Scanning Electron Microscopy, (Part I), Edited by O. Johari
and I. Corwin, ITT Research Institute, Chicago, pp. 381-402 (1975).
15. J. T. Thornthwaite, B. N. Thornthwaite, M. L. Cayer, M. A. Hart and R.
C. Leif; "A New Method of Preparing Cells for Critical Point Drying".
Scanning Electron Microscopy, (Part I), Edited by O. Johari and I. Corwin,
IIT Research Institute, Chicago, pp. 381-402 (1975).
16. R. C. Leif, S. Gall, L. A. Dunlap, C. Railey, S. B. Leif and R. M.
Zucker; "Centrifugal Cytology IV: The Preparation of Fixed Stained
Dispersions of Gynecological Cells". Acta Cytologica 19, pp. 159-168 (1975).
17. J. T. Thornthwaite and R. C. Leif; "Plaque Cytogram Assay I. Light and
Scanning Electron Microscopy of Immunocompetent Cells". J. Immunology, pp.
1897-1908 (1974).
18. M. A. Goldman and R. C. Leif; "Centrifugal Cytology II. Preliminary
Results on a Wet Chemical Method for Rendering Scanning Electron Microscopy
Samples Conductive and Observations on the Surface Morphology of Human
Erythrocytes and Ehrlich Ascites Cells". Proc. Nat. Acad. Sci. USA, 70, pp.
3599-3603 (1973).
19. R. C. Leif, H. N. Easter, Jr., R. L. Warters, R. A. Thomas, L. A.
Dunlap, and M. F. Austin; "Centrifugal Cytology I. A Quantitative Technique
for the Preparation of Glutaraldehyde-Fixed Cells for the Light and Scanning
Electron Microscope". J. Histochem. Cytochem. 19, pp. 203-215 (1970).
>Does CELL-TAk , SUPER FROST , or some other sticky stuff work?
Yes. See reference 16 R. C. Leif, et al. Acta Cytologica 19, pp. 159-168
(1975). Myers Albumin Fixative, which has similar properties to Elmer's
glue works the best. However, it is a source of background.
From: Robert C. Leif, Ph.D., PMIAC,
Vice President & Research Director
Newport Instruments
5648 Toyon Road
San Diego, CA 92115
Tel. & Fax (619) 582-0437
E-Mail rleif@crash.cts.com
=======================================================================
>
We use "Silane-prep" slides from Sigma (cat# S 4651), they are coated
with aminoalkylsilane. We sort directly to slides; we apply 15ul of 3%
BSA in PBS to the slide prior to sorting to prevent cell burst on
impact. We, however, use the slides for FISH, so you may not want to
use the same fixation proceedure. If you would like to try it: let
the slides dry overnight (room temp). We then apply a 3:1 mix of
Methanol:Acetic Acid. Allow it to dry, then apply again. Slides are
stored at -20. We had problems keeping cells on slides when we were
using uncoated slides; these slides have helped matters immensely.
Hope it helps you!
======================================================================
John Brandes M.D. Anderson Cancer Center
Phone: (713) 792-7640 Experimental Hematology
Fax: (713) 745-0887 1515 Holcombe Blvd.
e-mail:jbrandes@ix.netcom.com Box 081
Houston, TX, USA 77054
=======================================================================
`````````````````````````````````````````````````
Frederic I. Preffer
preffer@helix.mgh.harvard.edu
Department of Pathology- Cox5
100 Blossom St
Massachusetts General Hospital
Boston MA 02114
(617) 726-7481 {fax x2365}
~~~~~~~~~~~~~~~~~~~~~~~~~~~
|
|
|
|