18. Replace the plate cover and centrifuge 3 min at 900 x g, room temperature, with the brake on.

19. Remove the plate from the centrifuge and remove the supernatant by quickly turning the plate upside down so the supernatant is forcibly ejected from the plate. Next, while still holding the plate upside down, place it on an absorbent pad to pull the last remaining liquid from the wells.

20. Replace the cover and gently run the plate across a vortexer to loosen the cell pellet.

21. Using a transfer pipet, add 25 ^l/well of patient serum and any controls, using the plating format described in Table 6.16.5.

22. Replace the plate cover and mix the cell pools and serum by gently running the plate over a vortexer. Incubate the plate 30 min at 4°C.

Wash the plate

23. Remove the plate cover and use the repeat pipettor with 8-channel attachment to add 75 ^l cold flow wash buffer to each well. Replace the cover and vortex.

24. Add another 75 ^l to each well to complete the wash, but do not vortex at this point since splashover may occur.

25. Replace the plate cover and centrifuge 3 min at 900 x g with the brake on to pellet the cells.

Table 6.16.5 Plating Format for Flow Cell PRA
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