| 1. |
Collect
20 ml Blood in an EDTA containing vial |
| 2. |
Mix
it properly and keep at room temperature
|
| 3. |
Overlay
this blood sample on 2 ml Histopaque solution
in 25-ml sterile centrifuge tube. Centrifuge
the tube at 1500 rpm for 20 min |
| 4. |
Two
layers will be formed after centrifugation
|
| 5. |
Separate
out the upper layer of Histopaque with the
help of sterile pipette and collect the
lymphocyte ring in 2 ml sterile eppendorf
tube |
| 6. |
Microfuge
it for 1 min at the bottom of the eppendorf
tube lymphocytes sediment will be observed. |
| 7. |
Resuspend the sediment in 2-ml fresh RPMI-C
Media. |
| 8. |
Prepare
the serial dilutions of the drug of our
interest or to be tested in the range of
1ug/ml – 500 ug/ml |
| 9. |
Add
500 ul of the lymphocyte culture with the
help of sterile pipette / tips and add the
appropriate dilution of the drug to the
respective culture well along with control
without addition of any drug. |
| 10. |
10.
Incubate it in CO2 incubator at 37 degree
Celsius (5% CO2) |
| 11. |
After
incubation do the P-24 ELISA test for Control
and for Test |
| 12. |
Interpret
the results on the basis of difference in
between the Control O.D and Test O.D and
calculate the % Inhibition of the viral
growth in Test in comparison with Control. |
