LIFE09200604007 Tabish - Homi Bhabha National Institute
LIFE09200604007 Tabish - Homi Bhabha National Institute
LIFE09200604007 Tabish - Homi Bhabha National Institute
You also want an ePaper? Increase the reach of your titles
YUMPU automatically turns print PDFs into web optimized ePapers that Google loves.
Materials and Methods<br />
Immunophenotyping: 1x10 6 cells from LCLs were incubated separately with PE<br />
labeled primary mouse anti-CD3 (T cell marker), anti-CD19 (pan-B cell marker) and<br />
anti-CD56 (NK cell marker) antibodies for 1 h on ice (antibodies used in this<br />
experiment were kind gift from Dr. Shubhada Chiplunkar, Immunology department,<br />
ACTREC). The cells were washed and suspended in 500 µl FACS buffer. Further cells<br />
were passed through BD TM 1 ml 26G ½ syringe to break any cell aggregates or clumps<br />
and analyzed on Flow Cytometer (FACS Calibur, BD Biosciences, USA) at 488 nm<br />
excitation. A minimum of 10,000 events were analyzed for each sample. Cellular debris<br />
was removed by gating on Forward vs. Side Scatter. Data analysis was done using<br />
CellQuest Pro software (BD Biosciences, USA).<br />
3.4.2 Ploidy analysis of LCLs:<br />
DNA ploidy is defined as diploid DNA represented as single G0/G1 peak on a<br />
histogram of test sample corresponding to the same DNA content represented as single<br />
G0/G1 at the same position in the histogram of control sample. DNA ploidy was<br />
measured by calculating DNA index (DI) which is the ratio between the channel<br />
number of G0/G1 peak on histogram of the cell line to the channel number of G0/G1<br />
peak of control PBLs. DNA content of the cell is measured by labeling the cells with<br />
Propidium Iodide (PI). PI is a fluorescent intercalating agent which is when excited at<br />
488 nm fluoresces red. It is routinely used to stain DNA to evaluate cell cycle analysis<br />
and cell viability.<br />
Materials:<br />
1. 70% ethanol<br />
2. Propidium Iodide (0.4 mg/ml)<br />
3. RNase A (1 mg/ml)<br />
4. 1ml 26G ½ syringe<br />
5. FACS tubes<br />
Method:<br />
1x10 6 cells from LCLs and control PBLs were washed and suspended in 500<br />
µl PBS. Equal amount of 70% alcohol was added to the cells very slowly and drop<br />
wise. The cells were then mixed gently and were incubated for 10 min/ RT. Further<br />
Cells were centrifuged at 1500 rpm/ 10 min/ RT and were fixed in 500 µl 70% ethanol<br />
at 4 °C for 1 h. These fixed cells can be stored in 70% alcohol for a long time at 4 °C<br />
till further use. At the time of ploidy analyses cells were centrifuged at 1500 rpm/ 10<br />
56