12. Thirteen days after plating, change the medium to 200 μL of
stage 3 medium. Add MafA-11R protein at a final concentration of 1 μM.
13. Fifteen days after plating, change the medium to 200 μL of
stage 3 medium. Add MafA-11R protein at a final concentration of 1 μM.
3.7 C-Peptide
Secretion Assay
1. Wash the cells with 200 μL of low-glucose KRBH buffer once.
Then, add another 200 μL of low-glucose KRBH buffer and
incubate the cells for 1 h at 37
C in a CO 2 incubator to wash
out the residual medium (see Note 23).
2. Replace the buffer with 100 μL of fresh low-glucose KRBH
buffer and incubate for 1 h at 37
C in a CO 2 incubator.
Transfer 80 μL of the buffer incubated with the cells to a
1.5 mL tube (see Note 24) and then aspirate the residual buffer.
Keep the tube on ice.
3. Add 100 μL of high-glucose KRBH buffer and incubate for 1 h
at 37
C in a CO 2 incubator. Transfer 80 μL of the buffer
incubated with the cells to a 1.5 mL tube (see Note 25) and
then aspirate the residual buffer. Keep the tube on ice.
4. Add 20 μL of 1 N NaOH to the cells, lyse them by pipetting
and then transfer them to a 1.5 mL tube. Centrifuge at
12,000 Â g for 5 min at 4
C and then transfer the supernatant
to a new tube.
5. Measure the C-peptide levels in the buffers (low glucose and
high glucose) incubated with the cells after centrifugation at
3000 Â g for 5 min at 4
C (see Note 26) using a mouse
C-peptide ELISA kit (Shibayagi) (see Note 27).
6. Measure the total protein concentration of the supernatant of
the lysed cells with Bradford reagent (see Note 28).
7. Divide the C-peptide levels by the total protein content to
determine the secreted C-peptide as ng/protein (mg).
4 Notes
1. These plasmids are available upon request.
2. The 96-well plates with Ultra-Web synthetic surfaces have been
discontinued. Plates with polyamide net-like fibers can be substituted for them [7]. For information about polyamide
net-like fibers, please visit http://www.vilene.co.jp/.
3. The differentiation media supplemented with the factors
should be used within 2 days.
4. The range as 0.6~0.8 of OD600 may work.
Pancreatic Differentiation Through Protein Transduction
93
stage 3 medium. Add MafA-11R protein at a final concentration of 1 μM.
13. Fifteen days after plating, change the medium to 200 μL of
stage 3 medium. Add MafA-11R protein at a final concentration of 1 μM.
3.7 C-Peptide
Secretion Assay
1. Wash the cells with 200 μL of low-glucose KRBH buffer once.
Then, add another 200 μL of low-glucose KRBH buffer and
incubate the cells for 1 h at 37
C in a CO 2 incubator to wash
out the residual medium (see Note 23).
2. Replace the buffer with 100 μL of fresh low-glucose KRBH
buffer and incubate for 1 h at 37
C in a CO 2 incubator.
Transfer 80 μL of the buffer incubated with the cells to a
1.5 mL tube (see Note 24) and then aspirate the residual buffer.
Keep the tube on ice.
3. Add 100 μL of high-glucose KRBH buffer and incubate for 1 h
at 37
C in a CO 2 incubator. Transfer 80 μL of the buffer
incubated with the cells to a 1.5 mL tube (see Note 25) and
then aspirate the residual buffer. Keep the tube on ice.
4. Add 20 μL of 1 N NaOH to the cells, lyse them by pipetting
and then transfer them to a 1.5 mL tube. Centrifuge at
12,000 Â g for 5 min at 4
C and then transfer the supernatant
to a new tube.
5. Measure the C-peptide levels in the buffers (low glucose and
high glucose) incubated with the cells after centrifugation at
3000 Â g for 5 min at 4
C (see Note 26) using a mouse
C-peptide ELISA kit (Shibayagi) (see Note 27).
6. Measure the total protein concentration of the supernatant of
the lysed cells with Bradford reagent (see Note 28).
7. Divide the C-peptide levels by the total protein content to
determine the secreted C-peptide as ng/protein (mg).
4 Notes
1. These plasmids are available upon request.
2. The 96-well plates with Ultra-Web synthetic surfaces have been
discontinued. Plates with polyamide net-like fibers can be substituted for them [7]. For information about polyamide
net-like fibers, please visit http://www.vilene.co.jp/.
3. The differentiation media supplemented with the factors
should be used within 2 days.
4. The range as 0.6~0.8 of OD600 may work.
Pancreatic Differentiation Through Protein Transduction
93