Isolation of mononuclear cells by Ficoll Hypaque gradient separation

Equipment and reagents3

Method

1 Dilute blood from a buffy coat (30-40 ml of blood) 1:2 with Ca^/Mg2"1 -free PBS and mix well.

2 Place 30 ml of the blood/PBS mixture in a 50 ml conical centrifuge tube.

3 Slowly layer 15 ml of the Ficoll-Hypaque solution (density 1.077 g/ml) underneath by using a needle.

4 Centrifuge gradient for 30 min at room temperature at 280 g with no brake,

5 Using a sterile pipette, remove the upper layer that contains plasma and most of the platelets,

6 Using new pipette, recover the mononuclear cell layer and transfer this to another 50 ml conical tube.

7 Bring the volume to 50 ml by adding an appropriate amount of Ca2" /Mg2+-free PBS.

8 Centrifuge at room temperature for 5 min at 280 g with low brake.

9 Remove the supernatant by using a pipette and discard.

10 Vigorously resuspend the cell pellet in 50 ml of Ca^/Mg^-free PBS.

11 Centrifuge at room temperature for 10 min at 90 g.

12 Wash the cell pellet again (as described in step 9-11).

13 Resuspend the final cell pellet in 10 ml of RPMI1640 medium containing 15% FBS.

14 Count the viable cells by trypan blue exclusion,

'All reagents must be at room temperature before use since gradient density varies according to temperature.

h Peripheral blood human monocytes can be separated from heparinized blood samples or from buffy coats of normal bank donors with sodium citrate as an anticoagulant. Blood bank buffy coats are obtained after centrifugation of blood bank bag (original volume 400 ml) and removal of the upper layer of the content. The separated fraction (25-40 ml) contains more than 90% of the leukocytes of the blood donation, 10% of the erythrocytes, and 5% of the plasma.

Macrophages can be further purified from mononuclear cells by using adherence to different surfaces (see Protocols 1-4), Percoll gradient (see Protocol 9), or counterflow centrifugal elutriation (see Protocol 10).

10 cm long metal needles

Ficoll-Hypaque solution (Seromed, density 1.077 g/ml)

• RPMI 1640 medium containing 15% FBS

• Buffy coats or heparinized blood"

Ca2+/Mg2+-free PBS (see Protocol 1)

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