Primary human monocyte and macrophage culture

Human hPMBC isolation

1. For each tube of blood, prepare equal volume as there is blood of Ficoll into a 50mL Falcon tube. Note, try to process the blood onto Ficoll as soon as possible after the collection.

2. Dilute blood in equal volume of HBSS (i.e. 1:2 dilution).

3. Carefully overlay the diluted blood using a 10mL serological pipette by holding the 50mL Falcon tube containing the Ficoll at a 45º angle slowly pipette out so that there is minimal disturbance to the interface.

4. Centrifuge at 500g, 35min, 20ºC, breaks off.

5. Carefully draw of the upper layer containing the diluted plasma leaving the PBMC layer at the interface. Carefully transfer the PBMC to a new 15mL Falcon tube. Top up the remaining volume of the 15mL falcon with HBSS.

6. Centrifuge at 430g for 10 min at 20ºC.

7. Wash once more in 6mL of HBSS and centrifuge at 430g for 10min at 20ºC.

8. Resuspend the cell pellet in 2mL of PBS and take a small aliquot for counting on haemocytometer.

Preparing CD14 PE antibody

1. Count how many PBMCs per donor i.e. use 10uL of PE-conjugate per 10 x 10^6 cells and 100uL running buffer (1xPBS + 0.5% FBS + 2mM EDTA).

2. Add CD14 into Running Buffer and spin at max speed, 4C, 15min to remove aggregates.

Labelling and separation of CD14+ cells

1. After isolating hPBMC, wash in PBS and count. Spin at 430g, 5min, 4C.

2. Resuspend cells in spun CD14 PE in running buffer (10uL CD14 PE in 100uL running buffer per 10 x 10^6 cells).

3. Incubate for 60min in the dark at 4ºC fridge.

4. Wash cells twice (topping up with 5mL Running Buffer) at 430g, 5min, 4ºC.

5. Aspirate supernatant completely and resuspended in 80uL of running buffer per 10 x 10^6 cells.

6. Add 20uL of Anti-PE Microbeads per 10^7 total cells and mix well.

7. Incubate at 4ºC fridge for 15min.

8. Add 2mL of running buffer and centrifuge at 430g, 5min, 4ºC.

9. Aspirate supernatant completely and resuspend in 520uL buffer for up 10 x 10^7 cells. This is to aliquot 10uL out as PRE-separation, thus giving you a remaining ~500uL for separation. [Use this as a baseline to gauge on % CD14+ before enrichment].

10. Place LS column onto the magnetic field.

11. RINSE: To prepare the column for using add 3mL of running buffer and allow it to drip. The typical flow rate is 1.3-2.0mL/min.

12. Replace the collection tube with a new tube. This is to collect the unlabelled cells.

13. Add the entire cell suspension (i.e. the 500uL) onto the column.

14. Collect the unlabelled cells by washing the column with 3x 3mL running buffer (i.e. total of 9 min). NOTE: Only add new buffer when the columnreservoir is empty.

15. Remove the column from the separator and place it onto 15mL tube.

16. Pipette 5mL running buffer onto the column and immediately FLUSH out labelled cells by pushing the plunger provided into the column.

Further processing:

17. Centrifuge down the cells at 500xg for 6min at 4ºC.

18. Resuspend in 800uL of RPMI+20%FBS+P/S and took 10uL out for counting, then took another 10uL out flow.

19. Plate ~500,000 cells per 6-well plates.