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Isolation of lymphocytes from whole blood
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Materials required

 

  • 1X PBS Buffer
  • Histopaque 1077 medium
  • Freezing medium
  • Human blood
  • 20ml syringe with cannula
  • Heparinized green top tubes (4)
  • Centrifuge tubes(3)
  • Centrifuge
  • 1000 – 5000 microlitre procedure
  • Alcohol soaked cotton
  • 25ml glass pipette
  • 10ml glasspipette
  • Pipette pump
  • 50ml measuring cylinder
  • Waste beaker
  • 1.5ml cryovial (5) 
  • Vial rack
  • Cryobox
  • -80 °C freezer
  • Liquid nitrogen freezer

 

Procedure

 

 

  • 20 ml of blood is collected from the patient’s vein.
 
  • The collected blood is transferred to four 7ml heparinized green top tubes (Total: 20ml).
 
  • Pool all the blood from the heparinized tubes were pooled together into one 50ml blue cap tube.

 

  • Made 1:1 dilution of whole blood with 1X PBS

 

  • Carefully underlay 10ml of histopaque-1077. 20ml of diluted blood is transferred to a 50ml tube.

 

  • Immediately centrifuge at 1800rpm [700g] for 30 min in a cooling centrifuge (4°C).

 

  • Carefully remove the tube from the centrifuge, observing the three layers: top is clear supernatant, middle is opaque fluid containing the PBMC, and bottom is RBC. 

 

  • Carefully remove and discard the supernatant within 15 ml of the opaque layer of the PBMC. Quickly transfer the buffy coat layer of PBMC (around 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 1800rpm 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 1500rpm (500g) for 10 min in a cooling centrifuge (4°C) to remove platelets. 

 

  • Pour off and discard the supernatant. Resuspend the pellet in 5ml of the freezing medium (90%FBS+ 10%D MSO filter sterilized). 

 

  • Aliquot 1ml of cell suspension per vial (cryogenic vial). Store at 80°C overnight. Next day transfer in the Liquid Nitrogen cryofreezer. 

 

 

 

 

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by
Amrita CREATE (Center for Research in Advanced Technologies for Education),
VALUE (Virtual Amrita Laboratories Universalizing Education)
Amrita University, India 2009 - 2014
http://www.amrita.edu/create

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