University of Delhi

Materials

Materials

Preparation of ZnO/Ag2Onanocomposites

Tested individually

Dominance of regulatory transitional B cellsand lower memory B cells in HBsAgpositive compared to negative newborns

Post vaccination enhanced expression of activation marker CD69 and Chemokine receptor CCR5 on memory B cells in HBV positive newborns

Post-vaccination declination of Transitional B cell population and increase in memory B cells in HBV positive newborns

Increased Chemokine and Toll like receptor on T cells after vaccination in HBV positive newborns.

Vaccination improved the Chemokine receptor CCR1, CCR3, CCR9 and Toll like receptor TLR2, TLR4 and TLR9 expression in HBsAgpositive newborns compared to healthy newborns.

T cell frequencies(Post vaccination response)

Post-vaccination immune responses in newborns

IFN γ production by CD8 T cells upon stimulation with PMA and viral peptides

Decreased CD3 ζ chain expression on CD8 T cells in HBsAgpositive newborns

Phenotypic and Functional Characterization of CD8 T cells in cord blood

Cord Blood immune profiles of HBV positive newborns at birth(Cord blood vs. peripheral blood)

Pre-vaccination:Lower Chemokine and Toll like receptor expression in HBsAgpositive newborns:

T cell phenotypic distribution in HBsAgPositive, HBsAgNegative from HBsAg positive mothers and healthy newborns.

Clinical characteristics of the subjects

CD107a expression (marker of cytotoxicity)

Statistical Analysis

CFSE proliferation assay

CD107a Degranulation Assay

Flow cytometric analysis of cytokine production (IFN-γ)by intracellular staining

Flow cytometric analysis of intracellular CD3ζ chain expression

Cell surface and Intracellular staining and Flow Cytometric Analysis

Freezingor storageof cells

Cell Counting

Separation of Peripheral Blood Mononuclear Cells(PBMCs)from whole blood

Virological assessment: Quantitative RT-PCR detection of HBV DNA

Serological and Virological Assessment

HBV vaccination of the newborn

Sample Collection

Healthy Controls

Shrivastava, Shikha

To study the immune status of newborns to hepatitis B positive and negative mothers

Statistical analysis

NiSO4. 6H2O stock soloution

Nonidet-P40 (NP40) buffer for Ni-NTA pull down

Low-pH antibody elution buffer

Cytoskeleton extraction buffer (CSE)

Semi dry transfer buffer

GST elution buffer

Ni-NTA elution buffer

Lysis buffer for Ni-NTA and glutathione purification

1X TBST

1X TBS

1X PBS

Composition of buffers and solutions

GST-pull down

Surface plasmon resonance (SPR) measurements

Expression and purification of recombinant proteins

Immunofluorescence studies in SW80 cells for β-catenin under nickel induced condition

Luciferase assay in SW480 cells under nickel induced condition

Ni-NTA pull down

Stripping of Ni-NTA agarose beads

Nuclear and cytoplasmic fractionation

Large scale purification of BPN in CHO cells from pSecTag2 vector

Luciferase reporter assay

Immunoprecipitation and western Blotting

Immunofluorescence

Generation of polyclonal antibodies against GFP, APC and β-catenin

RNA Interference

Cell transfection

Secondary antibodies

Primary antibodies

Antibodies used in our study

Cell culture

General reagents and plastic wares

Medium and Serum

Animals

pGEX6P2-BPN (1-993 a.a)

pET30a-GFP-FL (GFP antigen)

pET30-b-APC Nde/Sca (1211-1495 a.a) (antigen for APC antibody)

pET30a-β-catenin-FL (Antigen for -catenin antibody)

GFP-β-cateninC-C (423-634 a.a) (ARM-C)

GFP-β-catenin-C-N (151-423 a.a) (ARM-N)

GFP-β-catenin-C (ARM)

GFP-β-catenin- C (634 -781 a.a)

GFP-β-catenin- a.a

GFP-β-catenin T41A mutant

Constructs made in the study

GFP-β-catenin-FL

National Centre for Cell Science

Tripathi Mukesh Mani

Novel roles of and#946; catenin and Nup358 in Wnt signaling