6 ml of Ficoll-Hypaque solution was added to a 25 ml bind-cap centrifuge tube, 9 ml of medium containing 2 × 107 cells was added on top, centrifuged, and live cells were collected from the junction site.

Operation method

Program 12.10 Enrichment of living cells

Principle

6 ml of Ficoll-Hypaque solution was added to a 25 ml bind-cap centrifuge tube, 9 ml of medium containing 2 × 107 cells was added on top, centrifuged, and live cells were collected from the junction site.

Materials and Instruments

Cell suspensions D-PBSA
Ficoll-Hypaque solution or other analogs
Centrifuge tubes or conventional containers Growth media Syringes Pipettes Cell counters Centrifuges

Move

1. Allow large tissue clumps in the suspension to settle.

2. Add 9 ml of cell suspension (with as few clumps as possible) on top of 6 ml of Ficoll (Ficoll/metrizoate to 1.077 g/ml) pantothenic acid solution in a wide-mouthed, clear, lidded centrifuge tube, such as a 25 ml sterile centrifuge tube or a Nunclon regular container (clear, shiny centrifuge tube or regular container), or a Corning 50 ml clear plastic tube with twice the volume of the above solution. Corning 50 ml clear plastic tubes, the volume of which should be twice the volume of the above solution.

3. 400 g (measured in the middle of the interface), centrifuge for 15 min .

4. Carefully remove the upper layer of liquid without damaging the interface.

5. Carefully collect the liquid at the interface with a syringe (with a blunt cannula or flat tip) or a Pasteur pipette or pipette.

6. Dilute the mixture to 20 ml with medium (e.g. DMEM/F12 with 10 % fetal bovine serum).

7. Centrifuge the mixture at 70 g for 10 min.

8. Remove the supernatant and resuspend the cell pellet with 5 ml of growth medium.

9 . Repeat steps 7 and 8 to make the cells free of density medium.

10. Count the cells using a blood counting plate or an electronic counter.

11. Inoculate into culture flasks for culture.

Caveat

Use human or other primate materials, not sharp needles or glass pasteurized pipettes.

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