Author: Hui-Yuen, Joyce; McAllister, Shane; Koganti, Siva; Hill, Erik; Bhaduri-McIntosh, Sumita
Title: Establishment of Epstein-Barr Virus Growth-transformed Lymphoblastoid Cell Lines Document date: 2011_11_8
ID: kss857n1_6
Snippet: 2. Underlay diluted blood with 15 ml Ficoll Hypaque lymphocyte separation medium. Centrifuge at 225 x g without brake at room temperature for 30 minutes. 3. Remove buffy coat and transfer into a new 50 ml conical tube. Raise volume to 50 ml with PBS and spin at 600 x g at room temperature for 10 minutes. 4. Pour off supernatant. Wash cells by resuspending pellet in 50 ml PBS and spinning at 600 x g at room temperature for 10 minutes. Wash twice m.....
Document: 2. Underlay diluted blood with 15 ml Ficoll Hypaque lymphocyte separation medium. Centrifuge at 225 x g without brake at room temperature for 30 minutes. 3. Remove buffy coat and transfer into a new 50 ml conical tube. Raise volume to 50 ml with PBS and spin at 600 x g at room temperature for 10 minutes. 4. Pour off supernatant. Wash cells by resuspending pellet in 50 ml PBS and spinning at 600 x g at room temperature for 10 minutes. Wash twice more in a similar manner. 5. Resuspend washed cells in 1 ml of complete RPMI. Use 5 μL of cells to prepare a 1/10 dilution in Trypan blue. Count live cells using a hemocytometer. 6. Adjust the volume using complete RPMI in a 25 cm 2 tissue culture flask to obtain a cell concentration of 2 x 10 6 /ml.
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