LIFE09200604007 Tabish - Homi Bhabha National Institute

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Synopsis Important note: For all cell characterisation and phenotypic assays freshly grown LCLs within 60 population doubling and with >90% viability were used. All biological waste was considered hazardous and discarded in 5% hypochlorite solution followed by safe disposal as per ACTREC bio-safety guidelines. 4. Characterization of LCL: Characterization of LCLs was done using standard techniques as described below. 4.1 Immunophenotyping: Cells (1x10 6 ) 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 used in this experiment were kind gift from Dr. Shubhada Chiplunkar and Dr. Naren Joshi, Immunology department, ACTREC). The cells were washed with FACS buffer and were passed through syringe to break any cell aggregates or clumps and analyzed on Flow Cytometer. A minimum of 10,000 events were analyzed for each sample. Cellular debris was removed by gating on Forward vs. Side Scatter. Statistical analysis was done using CellQuest Pro software. 4.2 Ploidy analysis of LCLs: DNA ploidy is defined as diploid DNA represented as single G0/G1 peak on a histogram corresponding to the same DNA content represented as single G0/G1 at the same position in the histogram of the control. 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. For ploidy analysis cells from LCLs and control PBLs from healthy volunteer were fixed in 70% ethanol. The cells were washed twice in PBS followed by incubation with Propidium Iodide and RNaseA. Cells were passed through syringe before acquisition to break any cell aggregates or clumps and fluorescence was acquired on flow Cytometer at 488nm excitation. Data were analyzed using ModFit LT V 2.0 software. 4.3 Expression and activity of ATM gene: RNA was isolated from cell lines and lymphocytes isolated from the same subjects by TRIzol extraction method. cDNA was synthesized from total RNA using Superscript First-Strand synthesis system by RT- PCR according to manufacturer‟s instructions. Expression of ATM gene was measured semi-quantitatively by RT-PCR using gene specific primers and β actin was used as 5
Synopsis loading control. PCR products were run on 2% agarose gel and stained with ethidium bromide. For one sample, activity of ATM gene was compared between the PBL and its corresponding cell line, by immunostaining for pATM. Both the cell line and isolated lymphocytes were irradiated with 4 Gy radiation dose so as to activate ATM in phosphorylated form. Unirradiated cell line and lymphocytes treated in the same way served as control. Following 30 min incubation cells were attached on the surface of Poly-L-lysine coated cover slips and fixed with 4% paraformaldehyde. Permeabilization was performed by addition of 0.3% TritonX-100 in PBS, followed by blocking in 3% BSA in PBST (PBS containing 0.1% Tween-20). Next the cells were incubated with pATM primary antibody at 1:150 dilution in PBST overnight at 4 o C. pATM was visualized as foci by using Alexa-546 secondary antibody at 1:200 dilution in PBST and nuclei were counterstained with DAPI. pATM appeared as red foci on DAPI stained nuclei. 4.4 Cell population doubling: 5x10 4 cells from LCLs were seeded for each time point in a 24 well plate with 1.5 ml of complete medium. Viable cell count was taken using Trypan blue dye exclusion method at different time points including 0, 12, 24, 36, 48, 72 and 96 h. For each time point four readings were taken. 5. Measurement of DNA damage and repair: LCLs were treated with γ-radiation to assess difference in the DNA repair capacity between MPN patient and control groups. 5.1 Immunofluorescence foci formation assay for γ-H2AX after γ-radiation exposure: 0.3x10 6 cells were irradiated with 2 Gy and 5 Gy γ-radiation in two sets using a Cobalt-60 isotope source (<strong>Bhabha</strong>tron-II) at room temperature. Unirradiated cells treated in the same way served as control. Following 30 min incubation (maximum γ-H2AX are seen after 30 min) from one set and 4 h incubation (time taken to repair the DNA) from the other set, cells were collected. Cells were attached, fixed, permeabilized and treated with blocking solution in the same way as described in 4.3. Cells were incubated with γ-H2AX primary antibody at 1:150 dilution in PBST overnight at 4 o C. γ-H2AX was visualized as foci by using Alexa-546 secondary antibody at 1:200 dilution in PBST and nuclei were counterstained with DAPI. γ-H2AX appeared as red foci on DAPI stained nuclei. 6
Page 1: Genotype-Molecular Phenotype Correl
Page 7 and 8: CONTENTS Page No Synopsis 1 List of
Page 9 and 10: Synopsis
Page 11 and 12: Synopsis Genotype-Molecular Phenoty
Page 13: Synopsis Materials and Methods: 1.
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
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Materials and Methods Source of che
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Materials and Methods Method: EBV-t
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Materials and Methods Immunophenoty
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Materials and Methods with 500 μl
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Materials and Methods cDNA it can b
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Materials and Methods Cobalt-60 iso
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Materials and Methods specific bind
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Materials and Methods h half of the
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Materials and Methods phenol and th
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Materials and Methods 7. Filter ste
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Materials and Methods viz. EXO-SAP
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Materials and Methods Table 2: List
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Materials and Methods SMAD6 AREG HM
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Materials and Methods 3.9 Effect of
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Materials and Methods fresh eppendo
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Materials and Methods Power SYBR Gr
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Results Objective 1 To generate EBV
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Results Fig. 8: Frequency of second
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Results Table 6: Demographics of he
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Results Fig. 10: Cell population do
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Results Fig. 12: Cell surface marke
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Results 4.1.5 Expression of ATM gen
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Results Objective 2 To compare the
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Results Fig. 17: Standardization of
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Results Fig. 19: Standardisation of
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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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