Download Pro TechEx – Isolation of Rosette Cells

Pro TechEx – Isolation of Rosette Cells
Teaching Kit
CAT.NO: OP-3710-10xp
0
Pro TechEx – Isolation of Rosette Cells
Teaching Kit
Introduction

Rosette is the French diminutive of rose. It may refer to

Rosette (design), a small flower design
Erythrocyte rosetting or E-rosetting is a phenomenon seen through a microscope where red
blood cells (erythrocytes) are arranged around a central cell to form a cluster that looks like
a flower. The red blood cells surrounding the cell form the petal, while the central cell forms
the stigma of the flower shape. This formation occurs due to an immunological reaction
between an epitope on the central cells surface and a receptor or antibody on a red blood
cell.
The immune system in higher organisms is composed of many lymphocytes subpopulations.
In the human, one subpopulation of lymphocytes interact in-vitro with unsensitized sheep
erythrocytes to form structures termed “Rosette”. Rosette consists of a central lymphocyte
surrounded by a cluster of attached sheep erythrocytes. The adherence of sheep red blood
cells (SRBC) to a lymphocyte results in a formation termed a Rosette. Rosettes are formed
by a small percentage of lymphocytes from peripheral blood of normal non-immunized
people. Its role as an immunological tool is still controversial, although inhibition of the
Rosette formation by anti- lymphocyte serum (ALS) has been shown to be a very sensitive
index of the immunosuppressive activity of various ALS preparations.
Principle
Human T- lymphocyte can be identified and counted by their ability to form rosettes with
SRBC. The T – Cells have surface receptors CD2 that binds with sheep RBC and forms
characteristic floral structure called Rosette were found under the microscope (E-Rosette).
These receptors were earlier called TH-Human-T lymphocyte in the peripheral lymphoid
organ bear receptor for SRBC on the surface.
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Materials Provided
Volume
S.No
Components
10Exps
Storage
1
Lymphosep
30ml
4°C
2
10X PBS
20ml
4°C
3
1.5ml tubes
10Nos
RT
4
15ml Falcon tubes
10Nos
RT
5
Pasteur pipette
10Nos
RT
6
Leishman stain
20ml
RT
7
Glass slide
5Nos
RT
8
Protocol
1
RT
Procedure
Preparation of 1X PBS: Dilute 1ml of 10X PBS with 9ml of Distilled water.
Lymphocyte Isolation
1. Heparinize the blood drawn from the peripheral vein by adding EDTA/Heparin, and
shake well for 10 min.
2. Dilute the Heparinized blood with the ratio of 1:2 using 1XPBS.
3. Take 3ml of lymphosep in 15ml falcon tube and add 3ml of Heparinized blood
sample and centrifuge at 2000rpm for 20 minutes.
4. After centrifugation remove the interphase peripheral blood lymphocytes using a
sterile Pasteur pipette and transfer it to another centrifuge tube.
5. Make up the volume to 5ml with 1X PBS and centrifuge at 2000rpm for 5 minutes.
6. Wash the pellet with 3ml 1XPBS and centrifuge at 2000rpm for 5 minutes. Suspend
the pure pellet in 2ml of 1X PBS and store at 4°C, mark as isolated lymphocytes.
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E-Rosette Isolation
1. Centrifuge the sheep blood cells (SRBC) 1ml with 4ml of 1XPBS Buffer at 5000rpm
for 2 minutes.
2. Discard the supernatant and wash the pellet with 4ml of 1X PBS(Repeat the step for
2 times) at 5000rpm for 2 minutes. Finally the SRBC pellet suspend with 4 ml of
1XPBS buffer.
3. In 1.5ml tube Pipette out 0.5ml of isolated lymphocytes and mix gently with 0.5ml
of SRBC. Keep the incubation at 37°C for one hour (To form a rosette cells).
4. After incubation keep it in an ice bath or refrigerate at 4°C for 5 minutes.
5. Gently transfer few drops of the Rosette cells to a clean glass slide after that add
few drops of leishman’s stain and keep it in a room temperature for air dry. After 5
minutes, gently wash with tap water.
6. Finally observe the Rosette cells under 40X objective lens under microscope.
Result and Observation
Lymphocytes surrounded by three or more SRBC which form a E-Rosette and appear as
floral like structure.
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