LIFE09200604007 Tabish - Homi Bhabha National Institute

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Materials and Methods added to the cells and placed in an incubator maintained at 37 C with 5% CO 2 . After 24 h medium containing viral supernatant was aspirated without disturbing the cells and fresh complete DMEM was added. Medium was changes as and when required by carefully aspirating the old medium and adding new medium without disturbing growing clumps of transformed cells. After 3-4 weeks of incubation rosette morphology of cells ascertained the transformed phenotype of PBLs. Cells were mixed thoroughly to break clumps before splitting to ensure multiclonal population. 3.4 Characterization of LCL Characterization of LCLs was done using standard techniques as described below. Cell lines were grown in complete DMEM (10% FBS, 200 mM Glutamine, 1x PenStrep) in T25 flasks routinely. For all genotypic and phenotypic characterization assays freshly grown LCLs within 60 population doubling and with >90% cell viability were used. Viability count was done by Trypan Blue Exclusion method where 10 µl of cell suspension was mixed with 10 µl of Trypan blue (Sigma). 10 µl of this mixture was loaded on Neubauer counting chamber and cell count was taken. Trypan blue selectively colour dead cells blue while live cells with intact membrane remain colourless. 3.4.1 Immunophenotyping: Materials: 1. Anti-CD3, anti-CD19 and anti-CD56 antibodies 2. FACS buffer (PBS containing 2% FBS and 0.02% Na-azide) 3. 1ml 26G ½ syringe 4. FACS tubes Method: Flow cytometry: Cell surface markers, microscopic particles inside the cells and DNA content can be efficiently analysed by suspending them in a stream of fluid passing through an electronic laser detection apparatus by a technique called flow cytometry. This technique allows studying the physical and chemical characteristics of thousands of cells in a very short span of time. Cellular components to be analysed are labelled with fluorescent conjugated antibodies or fluorescent dyes that are excited by a laser and produce signal that can be captured and evaluated to interpret the results. 55
Materials and Methods Immunophenotyping: 1x10 6 cells from LCLs were incubated separately with PE labeled primary mouse anti-CD3 (T cell marker), anti-CD19 (pan-B cell marker) and anti-CD56 (NK cell marker) antibodies for 1 h on ice (antibodies used in this experiment were kind gift from Dr. Shubhada Chiplunkar, Immunology department, ACTREC). The cells were washed and suspended in 500 µl FACS buffer. Further cells were passed through BD TM 1 ml 26G ½ syringe to break any cell aggregates or clumps and analyzed on Flow Cytometer (FACS Calibur, BD Biosciences, USA) at 488 nm excitation. A minimum of 10,000 events were analyzed for each sample. Cellular debris was removed by gating on Forward vs. Side Scatter. Data analysis was done using CellQuest Pro software (BD Biosciences, USA). 3.4.2 Ploidy analysis of LCLs: DNA ploidy is defined as diploid DNA represented as single G0/G1 peak on a histogram of test sample corresponding to the same DNA content represented as single G0/G1 at the same position in the histogram of control sample. DNA ploidy was measured by calculating DNA index (DI) which is the ratio between the channel number of G0/G1 peak on histogram of the cell line to the channel number of G0/G1 peak of control PBLs. DNA content of the cell is measured by labeling the cells with Propidium Iodide (PI). PI is a fluorescent intercalating agent which is when excited at 488 nm fluoresces red. It is routinely used to stain DNA to evaluate cell cycle analysis and cell viability. Materials: 1. 70% ethanol 2. Propidium Iodide (0.4 mg/ml) 3. RNase A (1 mg/ml) 4. 1ml 26G ½ syringe 5. FACS tubes Method: 1x10 6 cells from LCLs and control PBLs were washed and suspended in 500 µl PBS. Equal amount of 70% alcohol was added to the cells very slowly and drop wise. The cells were then mixed gently and were incubated for 10 min/ RT. Further Cells were centrifuged at 1500 rpm/ 10 min/ RT and were fixed in 500 µl 70% ethanol at 4 °C for 1 h. These fixed cells can be stored in 70% alcohol for a long time at 4 °C till further use. At the time of ploidy analyses cells were centrifuged at 1500 rpm/ 10 56
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Genotype-Molecular Phenotype Correl
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CONTENTS Page No Synopsis 1 List of
Page 9 and 10:
Synopsis
Page 11 and 12:
Synopsis Genotype-Molecular Phenoty
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Synopsis Materials and Methods: 1.
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Synopsis loading control. PCR produ
Page 17 and 18: Synopsis 7.1 Percent cell death aft
Page 19 and 20: Synopsis slides were then dried and
Page 21 and 22: Synopsis range 41.77% - 90.95% at 5
Page 23 and 24: Synopsis 5. Genotyping of genes: Ge
Page 25 and 26: Synopsis G2/M arrest and delaying e
Page 27 and 28: Synopsis may have an important bear
Page 29 and 30: Synopsis References: 1. Bobba, R.;
Page 31 and 32: Abbreviations MOPS : 3-(N-morpholin
Page 33 and 34: List of Figures Fig. 28: Percent re
Page 35 and 36: Chapter 1 Introduction
Page 37 and 38: Introduction In previous studies fr
Page 39 and 40: Introduction important carcinogenes
Page 41 and 42: Review of Literature The yet undeci
Page 43 and 44: Review of Literature Fig. 2: Incide
Page 45 and 46: Review of Literature sustained angi
Page 47 and 48: Review of Literature 2.6 Genotype p
Page 49 and 50: Review of Literature 2.7.2 Biologic
Page 51 and 52: Review of Literature Table 1: Steps
Page 53 and 54: Review of Literature future analysi
Page 55 and 56: Review of Literature Inefficient ce
Page 57 and 58: Review of Literature XRCC1 (X-ray r
Page 59 and 60: Review of Literature 2.9.2 Gene inv
Page 61 and 62: Review of Literature MPO gene polym
Page 63 and 64: Review of Literature important role
Page 65 and 66: Materials and Methods Source of che
Page 67: Materials and Methods Method: EBV-t
Page 71 and 72: Materials and Methods with 500 μl
Page 73 and 74: Materials and Methods cDNA it can b
Page 75 and 76: Materials and Methods Cobalt-60 iso
Page 77 and 78: Materials and Methods specific bind
Page 79 and 80: Materials and Methods h half of the
Page 81 and 82: Materials and Methods phenol and th
Page 83 and 84: Materials and Methods 7. Filter ste
Page 85 and 86: Materials and Methods viz. EXO-SAP
Page 88 and 89: Materials and Methods Table 2: List
Page 90 and 91: Materials and Methods SMAD6 AREG HM
Page 92 and 93: Materials and Methods 3.9 Effect of
Page 94 and 95: Materials and Methods fresh eppendo
Page 96 and 97: Materials and Methods Power SYBR Gr
Page 98 and 99: Results Objective 1 To generate EBV
Page 100 and 101: Results Fig. 8: Frequency of second
Page 102 and 103: Results Table 6: Demographics of he
Page 104 and 105: Results Fig. 10: Cell population do
Page 106 and 107: Results Fig. 12: Cell surface marke
Page 108 and 109: Results 4.1.5 Expression of ATM gen
Page 110 and 111: Results Objective 2 To compare the
Page 112 and 113: Results Fig. 17: Standardization of
Page 114 and 115: Results Fig. 19: Standardisation of
Page 116 and 117: Results The cells were incubated fu
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Results 4.2.3.1 Percent G2 delay af
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Results 4.2.3.2 Effect of BPDE expo
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Results Fig. 29: Comparison of perc
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Results Fig. 31: Percent cell death
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Results normalization with baseline
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Results 4.2.5.2 Real time PCR valid
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Results homozygous wild-type, heter
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Results Fig. 36b: Electrophoresis o
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Results Fig. 37a: Representative el
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Results Table 13: Consolidated geno
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Results LCL Genes NAT2(1) NAT2(2) N
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Results Fig. 38: Distribution of Ge
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Results Fig. 39: Various combinatio
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Results Table 14: Genotype-phenotyp
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Discussion Introduction Squamous ce
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Discussion stimulated lymphocytes w
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Discussion H2AX is currently the mo
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Discussion or G2/M phase arrest. As
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Discussion differential expression
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Discussion DNA repair genes and MPN
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Chapter 6 Summary and Conclusions
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Summary and Conclusions Statistical
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Bibliography 1. Dikshit, R.; Gupta,
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Bibliography 43. Lei, D.; Sturgis,
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Bibliography 76. Fry, R. C.; Svenss
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Bibliography 115. Kote-Jarai, Z.; M
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Bibliography 149. Hashibe, M.; Bren
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Bibliography 183. Bergamaschi, D.;
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Bibliography 216. Bondy, M. L.; Wan
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Indian J Med Res 135, June 2012, pp
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822 INDIAN J MED RES, JUNE 2012 mar
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824 INDIAN J MED RES, JUNE 2012 (h)
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826 INDIAN J MED RES, JUNE 2012 Tab
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828 INDIAN J MED RES, JUNE 2012 lik
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CASE REPORT Eben L. Rosenthal, MD,
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FIGURE 2. Chromosomal breakage anal
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FA along with ataxia telangiectasia
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LIFE09200604007 Tabish - Homi Bhabha National Institute

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