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Nile Blue Inducible Fluorescence of Tumour Cells

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Nile Blue Inducible Fluorescence of Tumour Cells INDUCIBLE FLUORESCENCE OF TUMOUR CELLS 1395 Nile Blue Inducible Fluorescence of Tumour Cells A. L. BASTOS and DANTE MARQUES Servio de Patologia Celular Calouste Gulbenkian, Instituto Portugues de Oncoldgia Francisco Centil, Lisboa 4, Portugal (Z. Naturforsdi. 27 b, 1395—1398 [1972] ; received June 6, 1972) Inducible fluorescence, tumor cells An oxazine dye, Nile Blue sulfate, induces a fluorescent reaction in cytoplasmic granules (NBIFG) of living or fixed tumour cells in the same manner as reported before for Thiazine dyes. The NBIFG correspond to phase contrast positive granules when cells are viewed by phase contrast microscopy. Fluorescence disappears from NBIFG in a matter of 2 —4 days and the bodies turn deep blue. These granules have a succinate dehydrogenase (SDG) activity and are negative for acid phosphatase, peroxidasic activity and porphyrin. The cytological findings support the assumption that Nile Blue sulfate forms a salt linkage with unsaturated fatty acids of NBIFG which also show an oxi-reductive activity. The molecular nature of the fluorophore (s) is (are) unknown. We have previously shown that thiazine dyes can Material and Methods induce a fluorescent reaction in cytoplasmic granu- les of tumour cells when freshly stained by these Tumour Cells dyes and examined by fluorescence microscopy 2. The staining procedures (supravital and in vivo 1 The fluorescent reaction is called primary when staining) were carried out using transplantable tumour cells of mice, Sarcoma 37 tumour cells and Ehrlich the granules are visualized immediately after the tumour cells growing in ascitic form. Cells were ob- staining; secondary, when the granules become tained from tumour-bearing mice 5 to 10 days after fluorescent only after exposure of the cells to light. cell transplantation. The experiments were carried out The primary reaction is observed only with a parti- while the tumour was maintained during 378 conse- cutive passages. cular batch of Toluidine Blue and the secondary reaction with all Thiazine dyes so far tested, in- Nile Blue Staining cluding samples obtained from several manufactu- A modification of L i 11 i e's3 technique for de- rers. monstration of fatty acids was used as follows: — The dye inducible fluorescent (DIF) granules are 1. Fix tumour cells in 4% formaldehyde saline at 4 C refractile, which makes their microscopic recogni- for 1 hours. tion easy (i. e. Interference microscopy); cytochemi- 2. Remove the excess fixative by cell centrifugation for cal reactions for lipids (Oil Red 0, Sudan Black 6-10 min at 1000 rpm. and reduction of osmium tetroxide) are positive. 3. Resuspend the cells in an aqueous solution of Nilft Blue (0.005% w/v) and stain for 30 min. Sudanophilia and osmiophilia usually indicate 4. Mount the cell sediment in glycerol. the presence of unsaturated fatty acids. Since the 5. Examine by fluorescence microscopy. presence of these highly oxidizable lipids could help to explain the cytochemical nature of the DIF Fixatives reaction, L i 11 i es Nile Blue procedure was used to To study the effect of fixation the following fluids demonstrate unsaturated fatty acids. were used: Neutral formalin at 10% and 15%, Car- noy's fluid, Alcohol-Ether, 2.5% glutaraldehyde in The observations now reported show that Nile cacodylate HCl buffer. Blue sulfate (samples from several manufacturers) also gave the DIF reaction in the same granules Control of pH, dye concentration, temperature and stained by Thiazine dyes, both in living and fixed staining time cells. a) pH: The cells were stained in Nile Blue solution at pH 0.9 -12. The pH was buffer adjusted with NaOH Requests for reprints should be sent to Dr. A. L. BASTOS, (saturated solution). Above pH 9 the colour of the Servio de Patologia Celular Calouste Gulbenkian, Instituto solution changes from blue to pink and precipitates. Portugues de Oncologia Francisco Gentil, Lisboa 4, Portu- b) Dye concentration and tempera- gal. ture: The dye concentration in the staining solution Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. 1396 A. L. BASTOS AND D. MARQUES was varied in tenfold dilutions from 0.5% to 0.00005% perchloric acid-mercapto ethylamine UV method for (w/v). The ratio of the cell volume to the volume of porphyrins 7. staining solution was 1 : 10. The temperature of the The above cytochemical preparations were then staining solution ranged from 0 °C to 100 °C. stained by Nile Blue sulfate to demonstrate the pre- c) Staining time: The staining times used sence of DIF granules and slides examined both by were als follows: - 0', T, 5', 10', 15', 30', 60', 2 hrs., light and fluorescence microscopy. 12 hrs., 24 hrs., 48 hrs. Nile Blue Compounds tested Results Nile Blue Sulfate [C.I. (913)51180] British Drug Houses, Poole, England, Batch N. 2868090; Matheson The fluorescence microscopic appearance of vi- Coleman & Bell, Norwood, Ohio, USA, Batch N. tally and supravitally stained and fixed tumour cells 481208; Chroma-Gesellschaft Schmid & Co., Stuttgart, are shown in Figs. 1,2* and 3. The Nile Blue indu- Germany, Batch unknown. All samples gave the fluo- rescent reaction. y Microscopic equipment _,40 UJ A Wild M20 microscope with a dual illumination O CC system for fluorescence, bright field and phase contrast UJ was used. It was fitted with Xenon lamp XBO 150w CL for fluorescence and a 12V/l00w quartziodine lamp for bright field and phase contrast. O <Z CL Fluorescence CD For Ultraviolet excitation — (c. 365 nm) the filter set consisted of a heat-absorbing filter KG 1 2, UG 1 3 4 O excitation filter . Red absorbing filter BG 38 and O 1 i GG 13 as the barrier filter. For Blue light excita- UJ tion — (c. 400 nm) a BG 12 3 excitation filter and C<O OG 1 3 barrier filter were used. £10 U. Cytochemistry O ca UJ In order to investigate the biological nature of DIF m granules, we employed the GOMORI lead phosphate ZD Z method 4 for lysosomes, peroxidactic activity in peroxi- 0 10 20 30 40 x somes, using 3 — 3' diamino benzidine (DBA) at pH 9 6, NUMBER OF FLUORESCENT GRANULES PER CELL succinodehydrogenase activity in mitochondria using a Graph I. Correlation of phase positive granules to fluorescent non-lipid soluble formazan forming salt-Nitro-BT 6 and bodies in 50 tumour cells. Fig. 1. Fluorescence microphotograph of ascitic Sarcoma 37. Figs. 6 a and 6 b. Fluorescence microphotograph of formalin Tumour cells vitally stained by Nile Blue. Bright green (ultra- fixed Nile Blue stained cells. Fluorescent emission disappears violet excitation) granules occur in the cytoplasm of almost in two days becoming the granules deep blue in bright field, every cell. Nucleus are non-fluorescent. 800 x. Fig. 6 b. 1200 x. Figs. 7 a and 7 b. Fluorescence microphotograph of an acid Fig. 2. Sarcoma 37 cells supravitally stained by Nile Blue. phosphatase preparation of Nile Blue stained tumour cells. Green colour emission of cytoplasmic granules as in vital The NBIFG do not show acid phosphatase activity although staining. 800 x. there are at least eight darker stained cells showing acid phos- phatase in bright field, Fig. 7 b. 1200 x. Fig. 4 a. Formalin fixed Sarcoma 37 tumour cell stained by Figs. 8 a and 8 b. Fluorescence microphotograph of peroxidac- Nile Blue and observed under bright field. No cytoplasmic tic activity in Sarcoma 37 tumour cells stained by Nile Blue. granules are seen. 2000 x. The NBIFG show no activity when the erythrocytes and neu- trophil have a positive reaction in the same preparation, Fig. 4 b. Phase contrast microphotograph of the cell shown Fig. 8 b. 1200 x. in Fig. 4 a. Note the presence of more than ten phase contrast Figs. 9 a and 9 b. Fluorescence microphotograph of five Sar- positive granules and four macro nucleolus. coma 37 tumour cells stained for succinic dehydrogenase and DIF reaction. Note the distribution of succinic dehydrogenase Fig. 5 a. Phase contrast microphotograph of formalin fixed activity in the same cells photographed in bright field, Fig. Nile Blue stained Sarcoma 37 tumour cells showing the pre- 9 b. 1200 x. sence of cytoplasmic granules. 1200 x. Figs. 10 a and 10 b. Diffuse cytoplasmic porphyrin emission Fig. 5 b. Fluorescence microphotograph of the same field as (red) of tumour cells which is weak when compared to the Fig. 5 a. Note the size, form and distribution of fluorescent reaction in erythrocytes (arrows). When these porphyrin pre- granules. parations of tumour cells are stained by Nile Blue (10 b) DIF granules are present showing yellow emission with blue light * Fig. 2 s. Tafel S. 1396 a. excitation. 1200 x. A. L. BASTOS and D. MARQUES. Nile Blue Inducible Fluorescence of Tumour Cells (S. 1395) Zeitschrift für Naturforschung 27 b, Seitel396 a Zeitschrift für Naturforschung 27 b. Seite 1396 b INDUCIBLE FLUORESCENCE OF TUMOUR CELLS 1397 V fluorescence microscopy. Graph 1 represents the cor- (* relation of phase-positive granules per cell to * * e fluorescent granules per cell. * A constant observation of Nile Blue stained O „ (pH 5) fixed cells is the disappearance of the # fluorescence in a matter of 2 — 4 days with the gra- * •' ' * * nules turning deep blue. § % Visual control of the preparations obtained i- > 0 under different staining conditions (Dye concentra- * * tion, temperature and staining time) showed that t fluorescence of NBIF granules (Excitation at 400) » was not affected by these variables.
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