Table 2 Protocol for isolation of proximal tubular cells

1.

Use the cell suspension obtained in Table 1.

2.

Add 20 μL of FcR blocking reagent.

3.

Mix well and incubate at 4°C for 10 minutes.

4.

Add 30 μL of anti-prominin-1 microbeads antibodies.

5.

Mix well and incubate at 4°C for 10 minutes.

6.

Add 10 μL of anti-prominin-1 APC antibodies.

7.

Mix well and incubate at 4°C for 5 minutes.

8.

Wash cells by adding 10 mL of dissociating buffer.

9.

Centrifuge at 500 g for 10 minutes.

10.

Aspirate the supernatant completely.

11.

Expand the cell pellet in 500 μL of dissociation buffer.

12.

Apply the solution to an LS column primed with 3 mL of buffer.

13.

Rinse the column with 3×3 mL of buffer, keeping the LS column away from the magnet.

14.

Collect the flow through solution and centrifuge at 500 g for 10 minutes.

15

Discard the supernatant and add 500 μL of buffer.

16.

Insert anew LS columns into an MACS separator magnet and prime it with3 mL of buffer.

17.

Apply the cell suspension obtained in 15.

18.

Wash the column with 3×3 mL of buffer.

19.

Remove the column from the separator and place it on a suitable collection tube.

20.

Flush the magnetically labeled cells with 5 mL of dissociation buffer.