LIFE09200604007 Tabish - Homi Bhabha National Institute

Materials and Methods
Method:
EBV-transformed B95-8 marmoset cell line was procured from National
Centre for Cell Science (NCCS) India to prepare EBV crude stock. B95-8 cell line was
revived and 0.5x10 6 cells/ml were seeded in complete RPMI-1640 in T25 flask. After 7
days confluent cultures of B95-8 appearing straw yellow in colour were lysed by freeze
thawing 3-4 times at -80 °C and 37 °C alternately. The lysed suspension was then
filtered through 0.22 μM filter to obtain EBV crude stock. The filtrate was aliquoted in
sterile 15 ml tubes and kept at 4 C for short term or -80 C for long-term storage.
3.3 Lymphoblastoid cell line preparation
LCLs are routinely developed by infecting PBLs with EBV which is known to
immortalize human resting B cells in-vitro giving rise to an actively proliferating cell
population.
Materials:
1. Ficoll-Hypaque
2. 24 well plate
3. EBV crude stock
4. DMEM (containing 15% FBS, 200 mM Glutamine, 1x PenStrep)
Method:
Ficoll-Hypaque: 250 ml, 9% w/v Ficoll was prepared in sterile distilled water. Solution
was heated at 45 °C to dissolve Ficoll completely. Simultaneously 100 ml, 33.3% w/v
Hypaque (Sodium diatrizoate) was also prepared in sterile distilled water and both the
solutions were mixed in the ratio of 24:10 i.e. 240 ml of Ficoll and 100 ml Hypaque, to
obtain final Ficoll-Hypaque gradient. Specific gravity of this gradient ranges from
1.076-1.078, which is ideal for separating PBLs from whole blood by centrifugation.
This is a simple and rapid method of purifying PBLs that takes advantage of the density
differences between mononuclear cells and other elements found in the blood sample.
Lymphoblastoid cell line preparation: For separation of PBLs approximately 3 ml of
blood was separated on a Ficoll-Hypaque gradient. Briefly, 3 ml of blood was mixed
with 3 ml PBS and carefully layered over 2.5 ml Ficoll-Hypaque in a 15 ml centrifuge
tube. Cells were centrifuged at 1500 rpm/ 20 min so as to obtain a ring of PBLs at
serum-Ficoll-Hypaque interface, while rest of the cells were collected at the bottom.
These PBLs were collected, washed with PBS and seeded in a sterile 24 well plate at a
density of 1.5-2x10 6 cells/ml in complete DMEM. EBV crude stock at 1:1 ratio was
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