LYMPHOCYTE CULTURE

BY: Ezhuthachan Mithu Mohanan (MSIWM043)

Cells circulating in body and part of immune system are known as lymphocytes. There are mainly two main types of lymphocytes, which includes B Cells and T cells. 

Peripheral Blood Lymphocyte culture: 

PBLC is a technique used in Cytogenetics used for karyotyping analysis. The most convenient method to study chromosomes and by using cell culture technique it can be helpful for clinical and research purposes.  The powerful mitogen for human T cell is PHA (Phytohaemagglutinin).PHA is a plant hormone, which is obtained from Red Kidney bean. The function of PHA is to bind to T Cell membrane, which stimulates metabolic activity.  It is important to obtain cells at metaphase plate, since the study of Chromosome is possible and also easy to stain the chromosome at metaphase stage. During metaphase chromosomes are attached to spindle fibres.

Procedure for PBLC

  • Cell culturing should be done in a laminar Hood to avoid contamination. 
  • 1 ml of Venous blood should be collected 
  • Take 7 ml of RPMI1640 ( reconstituted with 10% fetal calf serum) in sterile culture tube
  • Add 0.5 ml blood in culture tube
  • Add 0.1 ml of PHA ( Previously thawed) 
  • Incubate this tube in sterile CO2 Incubator at 37°C FOR 69 hours
  • Add 40 µL of Colchicine at 69:00 hours
  • Incubate for 30minutes at 37°C
  • After Incubation centrifuge at 2000 rpm for 15 minutes
  • Remove supernatant and add 5 ml of Hypotonic solution
  • Mix gently and Incubate in waterbath for 37°C for 20 minutes
  • Add ice cold fixative (1:3 acetomethanol) and flush thoroughly
  • Centrifuge at 2000rpm for 15 minutes and remove supernatant
  • Repeat this above two steps for 3-4 times
  • Mix final suspension of 1 ml of fixative and make drop preparation in cold wet slide
  • Fix on flame and stain with 4% Giemsa
  • Observe and Scan for well spread metaphase plate in microscope

Function of each reagent

  1. PHA: it binds to membrane of T Cell and stimulate metabolic activity and cell division
  2. Colchicine: Suppress mitosis and cell division, but there is no interruption of chromosome multiplication
  3. Hypotonic solution : Used for swelling of cells
  4. Aceto-methanol: Helps in hindering cells at metaphase stage 

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