Isolation of T Cells Using Rosetting Procedures

Isolation of T Cells Using Rosetting Procedures

1. A) ROSETTING WITH AET-TREATED SHEEP RED BLOOD CELLS(SRBC)

   • PREPARATION OF NEUROAMINIDASE-TREATED SRBC
     • 1.Place SRBC in Alsevers solution in centrifuge tube. Fill the tube with Hank Balanced Salt Solution (HBSS)
     • 2.Remove supernatant and resuspend cells (pellete) in the HBSS and centrifuge it again
     • 3.Add neuranimidase in PBS and resuspend the pellete with pipette (Incubate the tube)
     • 4.Discard the supernatant and resuspend cell again with HBSS (Repeat twice)
     • 5.Add complete RPMI-10 (media) to the pelleted neuraminidase-treated SRBC (store at 4°c)
   • FORMATION OF ROSETTES
     • 6.Transfer peripheral blood mononuclear cells (PBMC) in round-bottom centrifuge
     • 7.Remove supernatant and resuspend the cell in complete RPMI-10 media
     • 8.Add fetal calf serum (FCS) and neuroanimidase-treated SRBC
     • 9.Incubate the mixture at 37°c
     • 10.Place Ficoll-Hypaque solution in every mixture(PBMC/FCS/SRBC)
     • 11.Gently resuspend the mixture by tilting the tube to resuspend the tube (repeat step 10 & centrifuge)
     • 12.Remove 3/4 upper layer from centrifuge and transfer interphase layer to conical centrifuge tube
     • 13.Fill tube with HBSS & centrifuge it, resuspend cell and wash it.
   • LYSE OF SRBC
     • 14.Remove the remaining Ficoll-Hypaque solution. Separate E-rosette positive population from SRBC by hypotonic lysis.
     • 15.Centrifuge & remove the supernatant, resuspend(in complete RPMI-10) the cells in HBSS and repeat the wash.
     • 16.The isolated T cells now ready to be used

2. B)ROSETTING WITH NEURAMINIDASE-TREATED SHEEP RED BLOOD CELLS (SRBC)

   • 1.Add AET solution to packed. wash SRBC in centrifuge tube and incubate at 37°c
   • 2.Fill tube with cold PBS and centrifuge. Remove supernatant and resuspend cells in PBS (repeat 2x)
   • 3.Remove supernatant and add complete RPMI-10 to the pellet AET-treated SRBC (at last wash)
   • 4.Transfer mononuclear cells to round-bottom centrifuge tube, remove supernatant,resuspend in complete RPMI-10 to final concentration.
   • 5.Add FCS & SRBC. Centrifuge suspension and incubate cell mixture on ice.6. Follow step 10- 16 (basic protocol)

3. What is T-Lymphocyte

   • a type of lymphocyte (a subtype of white blood cells) that plays a central role in cell mediated immunity

4. What is rosetting techniques?

   • Used in immunology for the detection or purification of cells expressing a given surface ‘receptor’ of antigen