The document discusses various methods for evaluating anticancer activity and cell viability, including membrane integrity assays, functional assays, and DNA assays. Membrane integrity assays like trypan blue exclusion and LDH leakage are commonly used to determine cell viability by measuring intact cell membranes. Additional assays measure cellular functions, DNA integrity, morphology, and reproductive ability.

1. ANTICANCER ACTIVITY STUDIES: DIFFERENT MODELS Jesil Mathew. A, MCOPS, Manipal University

3. 2008 Estimated US Cancer Deaths* ONS=Other nervous system. Source: American Cancer Society, 2008. Men 294,120 Women 271,530 26% Lung & bronchus 15% Breast 9% Colon & rectum 6% Pancreas 6% Ovary 3% Non-Hodgkin lymphoma 3% Leukemia 3% Uterine corpus 2% Liver & intrahepatic bile duct 2% Brain/ONS 25% All other sites Lung & bronchus 31% Prostate 10% Colon & rectum 8% Pancreas 6% Liver & intrahepatic 4% bile duct Leukemia 4% Esophagus 4% Urinary bladder 3% Non-Hodgkin 3% lymphoma Kidney & renal pelvis 3% All other sites 24%

4. Change in the US Death Rates* by Cause, 1950 & 2005 * Age-adjusted to 2000 US standard population. Sources: 1950 Mortality Data - CDC/NCHS, NVSS, Mortality Revised. 2005 Mortality Data: US Mortality Data 2005, NCHS, Centers for Disease Control and Prevention, 2008. Heart Diseases Cerebrovascular Diseases Influenza & Pneumonia Cancer 1950 2005 Rate Per 100,000

12. There are many ways to die…..

13. Cell Viability • Functional assay • Membrane integrity assay • DNA labeling assay • Morphological assay • Reproductive assay

14. The major criteria employed in viability assay

15. Cell counting

17. Membrane integrity assays

19. Trypan blue  A stain which will only enter across the membranes of dead/non-viable cells. - Cause cancer in lab. animals - Appropriate precaution should be taken when handling trypan blue (use of extraction hood and gloves)  Dilution by trypan blue - Viable cells : small, round and refractive - Non-viable cells : swollen, larger, dark blue

21. Volume : 0.1mm 3 1 ml = 1 cm 3 = 1000 mm 3 1 mm 1 mm 0.1 mm

22. Dead cell

27. Automated trypan blue method for optimal cell viability determination www.innovatis.com

30. Result view of the image processing Marked viable and dead cells Viable cell Dead cell

31. Fig. a) Distribution histogram of the viable cell diameter for a human leukemia cell sample. b) Histogram of compactness for a human leukemia cell sample . The abscissa represents the ratio of cell circumstance to cell area normalized to the value of 1 for an ideal sphere (a) (b)

32. Vi-CELL TM CELL Viability Analyzer

34. Principle Trypan Blue dye Exclusion Methods

35. Run results

47. Schematic illustration of the principle of PI/FDA cell viability assay Intact cell – PI and FDA is added Fluorescein in intact cells ● FDA (Fluorescein diacetate ) ● PI (Propidium iodide) Plasma membrane is damaged ; fluorescein leaks out PI enters and strains nucleic acids

48. Example 1 ; Observation of cell death A group of hepatoma cells exposed to a diffusing wave of digitonin. Intact cells (green) are damaged by digitonin, loose the green fluorescence and acquire red fluorescence of PI.

49. Evaluate viability by examining the metabolic components that are necessary for cell growth , on the premise that cellular damage will inevitably result in the loss of ability to maintain and provide energy for metabolic function and growth. Functional assays

57. Example; NIH J2 3T3 cell in medium MTT solution elution Absorbance at 540 nm

59. metabolically active cell Water soluble

62. Example: MTT and XTT MTT XTT Jenny G., Mark H., Anna J., Inger K., Douglas Mc., Roland M., 2002. Evaluation of redox indicators and the use of digital scanners and spectrophotormeter for quantification of microbial growth in microplates. J. Micro. Methods. 50:63-73

65. Example; Monolayer culture Ref.:Journal of Orthopaedic Resarch19 (2001) ※ OP : Osteoporosis Crystal Violet - 60minutes Non-OP OP

66. Example; Microcarrier culture Rabbit oral mucosal cell cultured by microcarrier Cell counting

68. P-nitrophenyl phosphate + Acid phosphatase  Nitrophenol + HPO 4 -2

71. Procedure

77. Schematic diagram of [ 3 H]-TdR and BrdU

80. DNA labeling assay (using fluorescent probes assay)

81. Two types of nonviable cells

82. Two methods: Enzymatic DNA labeling and DNA-binding dye labeling

83. Ⅰ . Enzymatic DNA labeling dNTP dUTP Direct Indirect X Fluoresein, FITC, PE etc Biotin DIG Avidin conjugated with fluoresein, AP, POD Anti-DIG antibody conjugated with fluoresein, AP, POD

84. Fig. Immunostaining of apoptotic cells (dark brown) by TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL ) and peroxidase staining in rabbit endometrium

85. DNA-binding dyes: fluorochrome 1 RNase treatment is required 2 RNase treatment is required because PI could stain double strand RNA Ⅱ . DNA-binding dye labeling Dye Permeability via intact membrane Staining DNA RNA Acridine orange Yes Green Red-orange 1 Hoechst 33342 Yes Blue No Hoechst 33258 Yes Blue No DAPI (4,6-diamidino-2-phenylindole) Yes Bright blue No EtBr (Ethidium bromide) No Orange Slightly red 1 PI (Propidium iodide) No Red No 2

86. Pattern of dye staining according to color and chromatin morphology * late apoptosis is regarded as the stage of membrane fragmentation and secondary necrosis Dye Apoptosis Necrosis Early apoptosis Late apoptosis* Acridine orange Green Condensed Green Fragmented Green Diffuse, Intact Hoechst 33342 Blue Condensed Blue Fragmented Blue Diffuse, Intact Hoechst 33258 Blue Condensed Blue Fragmented Blue Diffuse, Intact DAPI Blue Condensed Blue Fragmented Blue Diffuse, Intact Ethidium bromide No (Orange, Condensed if permeabilized) Orange Fragmented Orange Diffuse, Intact Propidium iodide No (Red, Condensed if permeabilized) Red Fragmented Red Diffuse, Intact

87. Fig. In Hoechst 33258 / PI double staining, cells with blue intact nuclei were viable cells, whereas those with blue fragmented nuclei were early apoptotic cells. Cells with pink intact nuclei were necrotic cells, whereas cells with pink fragmented nuclei were late apoptotic cells. (blue against Hoechst33258, red against PI) Apoptotic(0%) Necrotic(10.5%) Apoptotic(85.2%) Necrotic(11.2%) Apoptotic(1.2%) Necrotic(92.5%)

88. Fig. DAPI staining of condensed nuclei of apoptotic cells

89. Acridine orange

92. Fig. Each part of quadrant statistics was observed under fluorescence microscopy. Live ECs preferentially expressed the green color of GS1 . Dead ECs are double stained by the green color of GS1-FITC and the red color of PI, which results in yellow . Dead cells except dead ECs are identified by only the red color of PI.

93. Morphological assay

95. Example; Morphological feature (Human skin keratinocyte) Fig. Morphological feature of (A) normal human skin keratinocyte, and differentiated human skin keratinocyte(B) . (A) (B)

96. Example; Morphological feature (Human skin fibroblasts) Fig. Morphological feature of (A) normal human skin fibroblasts, and aging human skin fibroblasts(B) . (A) (B)

97. Reproductive Assay

102. Example; Rat keratinocytes (A) (B) (C) (D) : colony , : Single cells Colony forming Non-colony forming 48 hr after subculture 6 days after subculture

105. In-vivo models