Biology Reference
In-Depth Information
13. IAA equilibration solution: Dissolve 0.125 g iodoacetamide
(IAA) and 1.8 g of urea in 5 mL of IPG equilibration buffer
(Serva). This recipe is designed for one strip. Adjust the amount
and volume according to the number of strips.
14. Ettan DALT
twelve
electrophoretic separation apparatus (GE
Healthcare).
15. Anode buffer: Dissolve the content of the anode buffer bag
(Serva) in 7.5 L of water.
16. Cathode buffer: Dissolve the powder in the cathode buffer bag
(Serva) in 5 L of water.
17. Low-melt agarose: Weigh 1 g of low-melt agarose (Quantum
Scientifi c, Queensland, Australia) and dissolve in 100 mL of
cathode buffer (see Subheading
2.4
, item 14) on a heated
magnetic stirrer (70°C).
18. ImageQuant TL image analysis software (GE Healthcare).
3. Methods
Once the CyDye is introduced to the sample, protect from light by
covering in aluminum foil.
1.
Thaw vials of cells (stored in liquid nitrogen) in a 37°C water
bath. Add 20 mL of culture media slowly in a dropwise fashion
then pellet cells by centrifugation at 400 ×
g
for 4 min at room
temperature. Remove supernatant, resuspend cells in 25 mL of
culture media, and transfer cells into a T75 culture fl ask.
Recover cells by incubating cells overnight in a humidifi ed
incubator with 4% CO
2
at 37°C.
3.1. Preparation
of Cells for LCL
2.
Transfer suspension cells into a new 50-mL Falcon tube and
centrifuge at 400 ×
g
for 4 min at room temperature. Remove
supernatant and resuspend cells in 20 mL of culture media.
Assess viability using trypan blue exclusion and hemocytome-
ter cell counting (
2
).
3.
Wash cells by resuspending the cell pellet in 5 mL of MACS
buffer. Centrifuge cells at 400 ×
g
for 4 min at room tempera-
ture. Remove supernatant and resuspend cells in 80 mL of
MACS buffer per 1 × 10
7
cells. Label CD3
+
cells by incubating
20 mL of CD3-MicroBeads per 1 × 10
7
cells in the dark for
15 min at 4°C.
4.
Place LD column in the MidiMACS separator and rinse column
with 2 mL of MACS buffer. Attach a pre-separation fi lter to the
top of the LD column and wet the fi lter with 0.5 mL of MACS
buffer and allow buffer to fl ow through into a waste tube.
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