By EDTA treatment

• 1:1 mixture of 10 mM EDTA in PBS pH 7.4 and RPMI 1640 medium containing 20% FBS (mixture A)

Protocol 6 continued

Method

1 Prepare a monolayer of adherent monocytes as described in Protocol 1.

2 Collect the non-adherent cell fraction as described in Protocol 5, step 2. For 75 cm2 flasks, add 10 ml of mixture A. Incubate cells for 15 min at 37°C.

3 Collect the detached cells (generally more than 90%) by gentle shaking of the flask and pipetting,

4 Transfer the cell suspension to 50 ml conical centrifuge tubes,

5 Centrifuge at 400 g for 5 min at 20°C.

6 Wash the cell pellet with 50 ml of RPMI1640 medium prior to subsequent analysis.

The efficiency of dislodgment of adhered cells by EDTA method can be improved at low temperature (4-20 °C). No mechanical scraping is generally required for further detachment under these conditions.

4.3 Recovery of adherent macrophages by lignocaine treatment

The local anaesthetic lignocaine, has been reported to provide reversible inhibition of cell monolayers with negligible toxicity, whereas other anaesthetics were found to be too toxic (56). A modification of the original protocol (57), which allowed greater recovery and viability, is described in Protocol 7.

Protocol 7

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