In the name of God

Summer School Influenza Unit, Pasteur Institute of Iran summer 2013

Isolate and culture of splenocyte Dr. Abbas Jamali

Summer School Influenza Unit, Pasteur Institute of Iran summer 2013 Introduction Cell culture Application?

Summer School Influenza Unit, Pasteur Institute of Iran summer 2013 Application of Animal Cell culture Analysis of cell signaling and differentiation Mechanisms of apoptosis and proliferation Gene expression and function Analysis of genetic defects and mutations Recombinant protein production (drug and vaccines) Production of crude vaccines (Rabies,polio,…) Identification of anti-cancer drugs(cytotoxicity test) Production of monoclonal antibodies Immunological assay

Primary cell culture suspension or adherent cultures

Summer School Influenza Unit, Pasteur Institute of Iran summer 2013 Spleen The spleen is situated in the left superior abdominal quadrant; it has a lengthened, oval, slightly curved shape. Spleen: an organ of predominantly lympho-erythropoietic function. A splenocyte can be any one of the different white blood cell types as long as it is situated in the spleen or purified from splenic tissue.spleen

Abdominal Quadrants Right Upper – Liver, Pancreas, Kidney, and Lung Right Lower – Appendix, Ureter, Bladder, Colon, and Gonads Left Upper – Heart, Spleen, Kidney, Stomach, and Lung Left Lower – Ureter, Bladder, Colon, and Gonads

Spleen Function The primary filtering element for the blood. Acts as a filter against foreign organisms that infect the blood stream. The site of red blood cell and platelet storage. Filters out old red blood cells and recycles them.

In order to study spleen cells (e.g. lymphocytes, granulocytes, other immune cells) can be a valuable source of either T or B lymphocytes. Summer School Influenza Unit, Pasteur Institute of Iran summer 2013 Splenocyte

Animal Models Summer School Influenza Unit, Pasteur Institute of Iran summer 2013 Body weight 25-40g F & 20-40g M Life span 3 M

Sacrificeing of mouse in a humane manner Summer School Influenza Unit, Pasteur Institute of Iran summer 2013 Chemical procedure Chloroform & ether Ketamine (overdose) ….. Physical procedure Dislocation of neck

Dissection of mouse spleen Summer School Influenza Unit, Pasteur Institute of Iran summer 2013

Dissection of mouse spleen Summer School Influenza Unit, Pasteur Institute of Iran summer 2013

Dissection of mouse spleen Summer School Influenza Unit, Pasteur Institute of Iran summer 2013

Splenocyte Isolation I II Summer School Influenza Unit, Pasteur Institute of Iran summer 2013

RBC Lysis Buffer Solution A – 8.29g NH4Cl – Add to 1L deionized water Solution B – 20.6g Tris-base – Add to 1L deionized water PH: mL of Solution A add to 10mL solution B pH solution to ; sterilize with autoclave; store at 4° Summer School Influenza Unit, Pasteur Institute of Iran summer 2013

Cell Counting Summer School Influenza Unit, Pasteur Institute of Iran summer 2013

Cell Counting Summer School Influenza Unit, Pasteur Institute of Iran summer 2013

Calculate Cell Concentration Cell/mL: Average number of cells in one large square x dilution factor* x 104

splenocyte Summer School Influenza Unit, Pasteur Institute of Iran summer 2013

Cell culture

Procedure

Summer School Influenza Unit, Pasteur Institute of Iran summer 2013

Early & Late Functions of Cellular Immunity IL-4IL-2 TNF  IFN  APC-T cell interactions Cytokine expression Proliferation/ Death Cyto- toxicity

Immunological Tests  Cytokine assay (ELISA)  Lymphocyte proliferative responses ( MTT )  CTL assay (Flow Cytometry ) Summer School Influenza Unit, Pasteur Institute of Iran summer 2013