Lymphocyte preperation_Animal Cell Culture.pptx

Lymphocyte preperation_Animal Cell Culture

1. Specialized techniques Lymphocyte Preparation

2. Introduction A lymphocyte is small white blood cells , usually 7 to 8 micrometers in length present in the blood . Lymphocytes are originated from lymphatic tissue throughout the body. It helps to protect the body from diseases, invasion of foreign bodies, tumors and infections.

3. Application of Lymphocytes The peripheral blood lymphocyte karyotyping provides information about chromosomal abnormalities. Production of cytokines. Production of mono- and polyclonal antibodies. Virus cultivation. Immunoclinical analysis.

4. Separation principle Typical process for lymphocyte separation is the density gradient centrifugation. Aqueous density media have been used with centrifugation to separate and isolate different blood cell types. Separation of lymphocytes from whole blood using HISTOPAQUE-1077 is based on a method first described by Boyum in 1968. The separation medium, HISTOPAQUE-1077 , is an aqueous solution of a high molecular weight polysaccharide and sodium diatrizoate , an iodinated nonionic compound, adjusted to a density of 1.077 ± 0.001. This medium facilitates rapid recovery of viable lymphocytes from small volumes of blood.  

5. Method 20 ml of blood is collected from a major vein   Transfer to four 7ml heparinized green top tubes   Pool all the blood from the heparinized tubes into a 50ml tube .   Make 1:1 dilution of whole blood with 1X PBS   Carefully add 10ml of histopaque-1077 to the diluted blood   Immediately centrifuge at 700g for 30 min in a cooling centrifuge (4°C )   Carefully remove the tube from the centrifuge, observing the three layers

6. Carefully remove and discard the supernatant within 15 ml of the opaque layer of the PBMC . Quickly transfer the buffy coat layer of PBMC (~ 5 ml) into a new 50ml tube.  Add 1x PBS to the PBMC suspension to obtain a final volume of 45 ml. Mix well and centrifuge at 700 g for 10 min in a cooling centrifuge (4°C). Pour off and discard the supernatant and combine 1 x PBS to obtain the final volume of 30 ml. Mix well and centrifuge at 500g for 10 min in a cooling centrifuge (4°C) to remove platelets. 

7. Pour off and discard the supernatant. Resuspend the pellet in 5ml of the freezing medium (90%FBS+ 10 % DMSO filter sterilized).  Aliquot 1ml of cell suspension per vial (cryogenic vial). Store at 80°C overnight. Next day transfer in the Liquid Nitrogen cryofreezer .