Purification of macrophages by counterflow centrifugal elutrlation CCE

Equipment and reagents

• CCE apparatus, including a variable-speed peristaltic pump regulated by a micrometer speed control and a pressure gauge

• Elutriation medium: Ca2+/Mgi+-free PBS containing 5% FBS

Method

1 Assemble the apparatus as shown in Figure 3.

2 With the rotor stationary, start the pump and set the speed such that about 40 ml/ min of the sterilizing agent (i.e. 70% ethanol, 6% hydrogen peroxide, or 0.2% diethyl pyrocarbonate) is drawn into the system (300-500 ml in total).

3 Rinse the apparatus successively with 500 ml of sterile distilled water and 500 ml of elutriation buffer.

4 Clear the elutriation chamber system of all air by adjusting the elutriation rotor speed to 750 r.p.m. and the peristaltic pump to a flow rate of 14 ml/min.

5 Increase the rotor speed to 2000 r.p.m. and adjust the flow rate to 6 ml/min.

6 When the CCE system has been calibrated to the above settings, inject the mononuclear cell suspension (4-5 ml in elutriation buffer) into the system with a sterile syringe connected to the three-way valve.

7 Turn off the pump to stop the flow of elutriation medium and allow the cells to enter the system,

8 Turn on the three-way valve at the completion of cell entry. Extreme caution must be used to prevent any air bubbles from entering the elutriation chamber, since this will create a back pressure that will cause fluid flow through the elutriation chamber to cease.

• Ficoll-Hypaque derived PBMC (Protocol 8)

• 70% ethanol, or 6% hydrogen peroxide, or 0.2% diethyl pyrocarbonate

• Sterile distilled water

Protocol 10 continued

9 Start to collect cell fractions in 50 ml aliquots in centrifuge conical tubes immediately after cell loading.

10 Gradually increase the flow rate to allow different cell populations to leave the elutriation chamber.

11 Collect 50 ml fraction in 50 ml centrifuge tubes.

12 Analyse each fraction to determine the cell type collected in each fraction.

13 At completion of the elution, wash the apparatus with sterile CaJ+/Mg2 ,-free PBS to eliminate residual cells and serum proteins, followed by 70% ethanol.

CENTRIFUGE

Elutriation medium

LAMINAR FLOW HOOD

Pump Pressure gauge

Pump Pressure gauge

Cell loading
Fraction collection

Figure 3 Schematic representation of the complete elutriation system. A diagrammatic representation of a complete elutriation system and of the different elutriation steps is Illustrated.

The first fractions collected will contain platelets and cell debris followed by erythrocytes eventually contaminating the mononuclear cells. Next, fractions of lymphocytes (pure lymphocyte fraction), followed by small monocytes and lymphocytes (intermediate monocyte fraction), and finally purified monocytes (purified monocyte fraction) will be obtained. This latter fraction will also contain small proportion of natural killer (NK) cells and large lymphocytes (about 2%).

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