LIFE09200604007 Tabish - Homi Bhabha National Institute

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822 INDIAN J MED RES, JUNE 2012 marker), anti-CD19 (pan-B cell marker) and anti- CD56 (NK cell marker) antibodies (BD Pharmingen, BD Biosciences, USA) for 45 min on ice. The cells were washed thrice with FACS buffer (PBS containing 1% FBS and 0.02% Na-azide) and incubated for 45 min on ice with secondary goat anti-mouse FITC antibody (antibodies used in this experiment were kind gift from Dr Shubhada Chiplunkar and Dr Naren Joshi, Immunology department, ACTREC). Cells were passed through BD TM 1ml 26G ½ syringe (BD, Singapore) 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. Statistical analysis was done using CELLQUEST software (BD Biosciences, USA). Ploidy analysis of LCLs: LCLs (1x10 6 ) and control PBLs (1x10 6 ) from healthy volunteer were fixed in 70 per cent ethanol at 4°C for 1 h. The cells were washed twice in PBS followed by incubation with propidium iodide (Sigma-Aldrich Co., USA) and RNaseA (Sigma- Aldrich Co., USA) for 30 min at 37°C. Fluorescence was acquired on Flow Cytometer at 488 nm excitation. Cells were passed through BD TM 1ml 26G ½ syringe before acquisition to break any cell aggregates or clumps and a minimum of 10,000 events were analyzed for each sample. Data were analyzed using ModFit LT V 2.0 software (BD Biosciences, USA). 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. Expression of ATM gene: RNA was isolated from cell lines and lymphocytes isolated from the same subjects by TRIzol (Invitrogen, Carlsbad, CA) extraction method. β actin PCR 18 was performed on isolated RNA to ensure any DNA contamination and samples were treated with DNase using DNA-free kit (Ambion, Austin, TX) wherever required. cDNA was synthesized using 3 μg of total RNA using Superscript First-Strand synthesis system by RT-PCR (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions. Expression of ATM gene was measured semi-quantitatively by RT- PCR using gene specific primers (Sigma-Aldrich Co., India; Forward 5’-TGTCATTACGTAGCTTCTCC-3’; Reverse 5’-GCTGAGTAATACGC AAATCC-3) and β actin was used as loading control. PCR products were run on 2 per cent agarose gel and stained with ethidium bromide. Cell population doubling: 5x10 4 cells from LCLs were seeded in a 24 well plate with 1.5 ml of complete medium. Viable cell count was taken using Trypan blue dye exclusion method 19 at different time points including 0, 12, 24, 36, 48, 72 and 96 h. For each time point four readings were taken. Results Establishment of LCLs and morphological analysis: Blood samples from MPN patients (n=24) and cancer free control individuals (n=13) were obtained and subjected to LCL preparation. The demographics of the patients and control individuals are shown in Table I. Considerable cell death of the PBLs was observed after 24 h post EBV infection; however, virus infection promoted B cells to re-populate the culture. The time taken for each LCL preparation varied. On an average culturing the cells 3-4 wk post infection was sufficient to produce >1 million cells. The average population doubling (PD) time of a few representative LCLs was found to be 24 h, ranging from 12 to 36 h (Fig. 1). The LCLs grew as clusters exhibiting typical rosette morphology in suspension cultures, but single cells were also observed having big nucleus and numerous vacuoles (Fig. 2a, 2b). EBV is known to specifically infect B cells allowing their growth in culture hence the transformed phenotype is expected to have homogeneous cell population, however, flow cytometric analysis revealed the presence of dual population (Fig. 2c). On the basis of morphology and granularity it was revealed that lower (R1) population represents mono cell suspension of interest and the other (R2) population probably represents fraction of cells that have spontaneously differentiated into smaller lymphoid cells with shrunken nucleus that ultimately undergo apoptosis during conventional cell culture thus representing diverse size and granularity 20 . Cell surface marker: Immunophenotyping was done using PBL as positive control for B (CD19), T (CD3) and NK (CD56) cells. Data confirmed that cells from representative randomly selected LCLs showed expression of typical B cell surface marker (CD19) while markers for T cell (CD3) and NK cells (CD56) were absent (Fig. 3), thus ascertaining the purity of growing cultures.
HUSSAIN et al: PREPARATION & CHARACTERIZATION OF LYMPHOBLASTOID CELL LINES 823 Table I. Demographics of subjects taken for lymphoblastoid cell line (LCL) preparation No. LCL Gender Age (yr) Category 1 st Cancer 2 nd Cancer 1 E245 M 52 Control - 2 E246 M 62 Control - 3 E247 M 52 Control - 4 E248 M 58 Control - 5 E249 M 46 Control - 6 E250 M 41 Control - 7 E301 M 24 Control - 8 E302 M 26 Control - 9 E303 M 25 Control - 10 E313 M 49 Control - 11 E314 M 61 Control - 12 E334 F 45 Control - 13 E335 F 65 Control - 14 E195 F 48 MPN Breast Hard palate 15 E242 M 45 MPN Tongue Hard palate 16 E252 M 38 MPN Buccal mucosa Buccal mucosa 17 E253 F 65 MPN Buccal mucosa Alveolous 18 E254 F 71 MPN Tongue Oesophagous 19 E264 M 71 MPN Lip Mouth 20 E265 M 48 MPN Larynx Tonsil 21 E270 M 60 MPN Alveolous Tongue 22 E305 F 64 MPN Retromolar trigone Alveolous 23 E306 M 55 MPN Larynx Hard palate 24 E307 M 49 MPN Larynx Nasal columella 25 E308 M 53 MPN Tongue Tongue 26 E311 F 63 MPN Tongue Buccal mucosa 27 E319 M 59 MPN Larynx Lung+ oesophagus 28 E320 F 45 MPN Right cheek Left cheek 29 E321 F 49 MPN Tongue Bronchus 30 E322 M 50 MPN Buccal mucosa Left lower lip+ right lower lip+ mouth 31 E325 F 43 MPN Buccal mucosa Alveolous 32 E326 M 75 MPN Supraglottis Oesophagous 33 E329 M 47 MPN GBS Alveolous+ buccal mucosa 34 E330 F 63 MPN Breast Mouth 35 E331 M 32 MPN AML Alveolous 36 E332 M 51 MPN Lung Parapharyngeal carcinoma 37 E333 F 55 MPN GBS GBS+ RMT+ alveolous MPN, multiple primary neoplasm; GBS, gingivobuccal sulcus; AML, acute myeloid leukaemia; RMT, retromolar trigone; M, Male; F, female
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Genotype-Molecular Phenotype Correl
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CONTENTS Page No Synopsis 1 List of
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Synopsis
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Synopsis Genotype-Molecular Phenoty
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Synopsis Materials and Methods: 1.
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Synopsis loading control. PCR produ
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Synopsis 7.1 Percent cell death aft
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Synopsis slides were then dried and
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Synopsis range 41.77% - 90.95% at 5
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Synopsis 5. Genotyping of genes: Ge
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Synopsis G2/M arrest and delaying e
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Synopsis may have an important bear
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Synopsis References: 1. Bobba, R.;
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Abbreviations MOPS : 3-(N-morpholin
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List of Figures Fig. 28: Percent re
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Chapter 1 Introduction
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Introduction In previous studies fr
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Introduction important carcinogenes
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Review of Literature The yet undeci
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Review of Literature Fig. 2: Incide
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Review of Literature sustained angi
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Review of Literature 2.6 Genotype p
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Review of Literature 2.7.2 Biologic
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Review of Literature Table 1: Steps
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Review of Literature future analysi
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Review of Literature Inefficient ce
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Review of Literature XRCC1 (X-ray r
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Review of Literature 2.9.2 Gene inv
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Review of Literature MPO gene polym
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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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Page 130 and 131: Results homozygous wild-type, heter
Page 132 and 133: Results Fig. 36b: Electrophoresis o
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Page 136 and 137: Results Table 13: Consolidated geno
Page 138 and 139: Results LCL Genes NAT2(1) NAT2(2) N
Page 140 and 141: Results Fig. 38: Distribution of Ge
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Page 144 and 145: Results Table 14: Genotype-phenotyp
Page 146 and 147: Discussion Introduction Squamous ce
Page 148 and 149: Discussion stimulated lymphocytes w
Page 150 and 151: Discussion H2AX is currently the mo
Page 152 and 153: Discussion or G2/M phase arrest. As
Page 154 and 155: Discussion differential expression
Page 156 and 157: Discussion DNA repair genes and MPN
Page 158 and 159: Chapter 6 Summary and Conclusions
Page 160 and 161: Summary and Conclusions Statistical
Page 162 and 163: Bibliography 1. Dikshit, R.; Gupta,
Page 164 and 165: Bibliography 43. Lei, D.; Sturgis,
Page 166 and 167: Bibliography 76. Fry, R. C.; Svenss
Page 168 and 169: Bibliography 115. Kote-Jarai, Z.; M
Page 170 and 171: Bibliography 149. Hashibe, M.; Bren
Page 172 and 173: Bibliography 183. Bergamaschi, D.;
Page 174 and 175: Bibliography 216. Bondy, M. L.; Wan
Page 176 and 177: Indian J Med Res 135, June 2012, pp
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Page 186 and 187: CASE REPORT Eben L. Rosenthal, MD,
Page 188 and 189: FIGURE 2. Chromosomal breakage anal
Page 190 and 191: FA along with ataxia telangiectasia
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