- Email: [email protected]
Fluorescent probes for imaging and flow cytometry in living cells
Biol. Cell (1991),73 NUCLEOLAR ORGANISER REGIONS (NORs) IN CYTOLOGIC IMPRINTS OF NON-HODGKIN'S LYMPHOMAS. A COMPARATIVE STUDY WiTH K! 67 EVALUATED BY IMMUNOHISTOCHEMISTRY AND FLOW CYTOMETRY ~ 1 , 2 GUILLAUD pl, LE M A R C H A D O U R 1:4, JACOB MC3,SOTTO MF2,SEIGNEURINDI,SOTTO Jj 2j,~ FN M4 i-Equipe de Cytologic Quantitative, 2-Laboratoire de Recherche en immanoputhologie Tumorule-LR !!, 3-1abor~roire dTrnnumologie. CTS. 4-Service d'Anatomie Pmhologique. Facuh# de Mddecine de Grenoble, 38700l a D'onche , France. Expression of two markers for assesing"cellular ixoliferation were investigatedin differenttypes of non-Hodgkin's lymphomas(NHL):NORs in cytologic imprints and Ki-67 antigen by immunohistochemismj and flow cytommry.$0 consecutifs cases of NHL were examined: 23 lowgrade and 27 high glade lymphomas as def'med by Kiel's classification; 21 Class I, 2 Class 2. 27 Class 3 as det'med by Grenobles cytological classification .The Ki-67 staining was quantitated by flow cytomemj and histological methods using APAAP preparations : Ki-67 positive cell enumerauon was cmieo out on a minimum $00 cells (at a 400 x maliniflcation).The NORs were marked by one seen silve~ staininz technique (AII-NORs) on cytologic imprints and wire examined by compumized system of imalie analysis SAMBA 200S (TrrN).Stadstical analyses snow an excellent posidve correlations between the Ki-67 score and NORs expresston: NORs NORs NORs/nucleaz number area ar~a ratio
m0.709
Histological evaluation
m0,816 p=O,O00l
Flow ¢ytomeay
r=0,63 r=0.q38 p-O,O001 p-O.O01$
p.~,O00I
r=0,73
p=O,O00l
r=0.449 p=0,01
b Asigniflcant cormlmion existed betweenthe Ki-67 scoreas dmmnined y PlOW c y t o m ~ and by histological enumeration on histological sections, ( r ~ 0.$46..p m 0.007). There were significant correlations oetween N.uRs.expresston (number,surface,NORa surface/nuclear surface ratio) ima ntstological (low gradsJdgh glade) as weU as cytological (class I and classes2-3) classification. NORs surface parameters seem to play an important role in distinguishing between high and low grade malignancy in cylological imprints of non Hodgkin's lymphomas.
FLUORESCENT PROBES FOR IMAGING ANO FLOW CYTOMETRY IN UVING CELLS _HAUGLANORichard P., ZHANG Yu.zhong, HUANG Zhljian, NALEWAYJohn, WELLS Slim, OLSON Nil|, YOU Wetmtn, LARISON Karan, KANG Hie Chol, KUHN Michael Molecular Probes, Inc., Eugene OR 9740g Oelermlnlng me~bollc phy|Iologleal |tats of living cells has been facilitated by development of in vivo fluorescent probes for measuring intracslluiar lone, enzymes, gang expression and other properties. This talk will Include several regent developments made In our laboratories, including: 1. Impmvtng cellular retention of fluorescent products formed by enzymalic action on synthetic substretes, palticulefly by forming fluorescent products that become membrenQ.bound, that react with intracellular components, thai form fluorescent precipitates, that react to form impermeant products or that bind to intrecellula¢ organelias, 2. New ion indicators, pmlicuiady for pH and calcium. 3. Improved methods for measuring cell viability and for cell tracing, 4. Site-selecUveprobes Ihat bind to cell receptors, membranes, ONA and RNA. In addition, new hlstochemloal and dlagno|tio applications of these new probes will be mentioned. This research supported by NIH grants R44 GM37347 and R44 GM3egII? to RPH.
46a
EVALUATION OF THE NEW FLUORESCENT PROBES FOR ROUTINE THREE COLOR CELL SURFACE FLOW C¥TOMETRIC ANALYSIS USING A SINGLE LASER. LOUISE Anne, JOUIN H~l~ne. Station de Cytofluorom~trie, D~partement d ' Im munologle, Institut Pasteur, 75724 PARIS C£DEX 15. Since the extensive use of simultaneous multiparametric immunofluorescence analysis b 7 flow cytometry, specially in the phenotyping of perlpheral blood lymphocytes, several fluorochromes h a s been d e v e l o p p e d t o p e r f o r m r o u t i , n e l y t h r e e c o l o r a n a l y s i s . These new f l u o r e s c e n t p r o b e s can be u s e d i n c o n j u n c t i o n w i t h FITC and P h y c o e r y t h r i n , r e q u i r i n g a f l o w c y tometer equipped with three fluorescence det e c t o r s , one l a s e r ( 4 8 8 nm), f o u r e l e c t r o n i c compensation netwoks, logarithmic amplifiers, and a p p r o p r i a t e t h r e e c o l o r f i l t e r s a r r a n g e ments, We have compared the results obtained using StregtavLdines conjugated with the different available new fluorochromes for routine three color cell surface analysis. We have tested five Streptavidines: -StreptavidLne-C¥-CHROME (Pharmingen) -Streptavidine-DUOCHROME (Becton D i c k i n s o n ) -Streptavidine-RED 613 (Immunoselect) -Streptavidine-TAND£M (Southern Inc) -Streptavidine-TRI-COLOR (Caltag) For each probe we have s t u d i e d : -the signal, -the compensation setting, -the price of one test.
m0.709
Histological evaluation
m0,816 p=O,O00l
Flow ¢ytomeay
r=0,63 r=0.q38 p-O,O001 p-O.O01$
p.~,O00I
r=0,73
p=O,O00l
r=0.449 p=0,01
b Asigniflcant cormlmion existed betweenthe Ki-67 scoreas dmmnined y PlOW c y t o m ~ and by histological enumeration on histological sections, ( r ~ 0.$46..p m 0.007). There were significant correlations oetween N.uRs.expresston (number,surface,NORa surface/nuclear surface ratio) ima ntstological (low gradsJdgh glade) as weU as cytological (class I and classes2-3) classification. NORs surface parameters seem to play an important role in distinguishing between high and low grade malignancy in cylological imprints of non Hodgkin's lymphomas.
FLUORESCENT PROBES FOR IMAGING ANO FLOW CYTOMETRY IN UVING CELLS _HAUGLANORichard P., ZHANG Yu.zhong, HUANG Zhljian, NALEWAYJohn, WELLS Slim, OLSON Nil|, YOU Wetmtn, LARISON Karan, KANG Hie Chol, KUHN Michael Molecular Probes, Inc., Eugene OR 9740g Oelermlnlng me~bollc phy|Iologleal |tats of living cells has been facilitated by development of in vivo fluorescent probes for measuring intracslluiar lone, enzymes, gang expression and other properties. This talk will Include several regent developments made In our laboratories, including: 1. Impmvtng cellular retention of fluorescent products formed by enzymalic action on synthetic substretes, palticulefly by forming fluorescent products that become membrenQ.bound, that react with intracellular components, thai form fluorescent precipitates, that react to form impermeant products or that bind to intrecellula¢ organelias, 2. New ion indicators, pmlicuiady for pH and calcium. 3. Improved methods for measuring cell viability and for cell tracing, 4. Site-selecUveprobes Ihat bind to cell receptors, membranes, ONA and RNA. In addition, new hlstochemloal and dlagno|tio applications of these new probes will be mentioned. This research supported by NIH grants R44 GM37347 and R44 GM3egII? to RPH.
46a
EVALUATION OF THE NEW FLUORESCENT PROBES FOR ROUTINE THREE COLOR CELL SURFACE FLOW C¥TOMETRIC ANALYSIS USING A SINGLE LASER. LOUISE Anne, JOUIN H~l~ne. Station de Cytofluorom~trie, D~partement d ' Im munologle, Institut Pasteur, 75724 PARIS C£DEX 15. Since the extensive use of simultaneous multiparametric immunofluorescence analysis b 7 flow cytometry, specially in the phenotyping of perlpheral blood lymphocytes, several fluorochromes h a s been d e v e l o p p e d t o p e r f o r m r o u t i , n e l y t h r e e c o l o r a n a l y s i s . These new f l u o r e s c e n t p r o b e s can be u s e d i n c o n j u n c t i o n w i t h FITC and P h y c o e r y t h r i n , r e q u i r i n g a f l o w c y tometer equipped with three fluorescence det e c t o r s , one l a s e r ( 4 8 8 nm), f o u r e l e c t r o n i c compensation netwoks, logarithmic amplifiers, and a p p r o p r i a t e t h r e e c o l o r f i l t e r s a r r a n g e ments, We have compared the results obtained using StregtavLdines conjugated with the different available new fluorochromes for routine three color cell surface analysis. We have tested five Streptavidines: -StreptavidLne-C¥-CHROME (Pharmingen) -Streptavidine-DUOCHROME (Becton D i c k i n s o n ) -Streptavidine-RED 613 (Immunoselect) -Streptavidine-TAND£M (Southern Inc) -Streptavidine-TRI-COLOR (Caltag) For each probe we have s t u d i e d : -the signal, -the compensation setting, -the price of one test.