Evaluating the Efficacy of Subcellular Fractionation of Blast Cells Using Live Cell Labeling and 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 325

5. Wash cells by adding 2 mL of MACS buffer per 1 × 10 7 cells.

Spin cells at 400 × g for 4 min at room temperature. Remove
supernatant and resuspend up to 1.25 × 10 8 cells in 500 mL of
MACS buffer.

6. Add cell suspension to the pre-separation fi lter and allow cells

to fl ow through to the LD column to a new collection tube.
Rinse the fi lter and column with 1 mL of MACS buffer. Repeat
rinsing once. Dispose of the column and fi lter.

7. Make the volume of cells up to 5 mL and perform viability cell

count using trypan blue. Spin cells at 400 × g for 4 min at room
temperature. Remove supernatant and resuspend cells in 80 mL
of MACS buffer per 1 × 10 7 cells. Add 20 mL of CD19-
MicroBeads per 1 × 10 7 cells and incubate 15 min at 4°C pro-
tected from light.

8. Prepare LD column and pre-separation fi lter (see Subheading 3.1 ,

step 4).

9. Wash cells by adding 2 mL of MACS buffer per 1 × 10 7 cells.

Spin cells at 400 × g for 4 min at room temperature. Remove
supernatant and resuspend up to 1.25 × 10 8 cells in 500 mL of
MACS buffer.
10. Add cell suspension to the pre-separation fi lter and allow cells
to fl ow through to the LD column to a new collection tube.
Rinse the fi lter and column with 1 mL of MACS buffer. Repeat
column rinsing once. Dispose of the column and fi lter.
11. Make up the volume of cells to 5 mL and perform viability cell
count using trypan blue.

1. Wash 1 × 10 7 cells in 10 mL of PBS. Spin cells at 400 × g for
4 min at room temperature. Repeat wash step twice. After the
last wash, resuspend cells in 100 mL of binding buffer and trans-
fer cell suspension to a 1.5-mL microcentrifuge tube. Add
600 pmol of Cy3 to the cells (see Note 3). Mix well by thorough
pipetting and centrifuge briefl y in a microcentrifuge. Leave on
ice for 30 min in the dark. Quench the labeling reaction by add-
ing 1 mL of 10 mM lysine. Mix by pipetting and spin briefl y in a
bench-top centrifuge. Leave for 10 min on ice in the dark.
2. Wash cells in 1 mL of PBS. Spin cells at 400 × g for 2 min at room
temperature. Repeat twice. Resuspend cells in 100 mL of PBS.

3.2. CyDye DIGE Fluor
Minimal Labeling
of Blast Cells

3. To evaluate the consistency and effectiveness of LCL using 1D

SDS PAGE, transfer 5 mL of cell suspension into a tube and
add 13 mL of 2× LDS sample buffer to lyse the cells. Mechanically
break up the DNA/RNA with a needle and syringe. Sonicate
sample in ice-cold water for 15 min. Spin tube at 10,000 × g for
10 min. Transfer supernatant into a new microcentrifuge tube.
Add 2 mL of 0.5 M DTT into the cell lysate to reduce disulfi de