MDDC generation from monocytes

(CD14+ positive selection)

Day 0:
1- Cut the bottom end of cone with scalpel/scissors. Balance over a 50ml falcon tube. Cut top
end of the tube and allow the cone to empty.
2- Once the cone is empty, wash through with PBS. Dispose the cone in the blue box, the scalpel
in a sharps bin.
3- Top up the blood to 50ml with PBS.
4- Fill two new falcon tubes with 12.5ml Lymphoprep.
5- Using a plastic pasteur pipette layer 25ml of blood onto the Lymphoprep.
6- Centrifuge 20min at 800g, RT, accelerator=5, brake=1.
7- Collect the lymphocyte layer and transfer in a new falcon tube. If the volume is more >5ml
divide in two (in order to dilute any lymphoprep sufficiently).
8- Fill each falcon to 50ml with PBS. Centrifuge 5min, 1500rpm, RT.
9- Discard the supernatant and wash the pellet 3x more in 50ml PBS. The supernatant should
become clearer each time, due to loss of platelets.
10- Combine the pellets resuspending in complete RPMI + 10% FBS, wash and count.
11- Take 2x106 cells for FACS staining. Stain with CD14/CD209/CD163/CD1a.

Magnetic bead labelling and separation in LS columns (max 108 labelled cells and 109 total cells):

12- Centrifuge cell suspension at 300g for 10 min, then aspirate supernatant completely.
13- Resuspend in 80l MACS buffer (2mM EDTA, 0.5% BSA, in PBS) per 107 total cells.
14- Add 16l of CD14 MicroBeads per 107 total cells.
15- Mix well and incubate for 15 minutes in the fridge.
16- Wash cells in MACS buffer (300g for 10 minutes). In the meantime, place LS column in the
magnetic field and rinse with MACS buffer to hydrate (3ml).
17- Resuspend up to 108 cells in 500l buffer.
18- Apply cell suspension onto the column.
19- Wash column (3 washes x 3ml buffer) and collect the unlabelled cell fraction.
20- Remove column from support, place it on collection tube, pipette 5ml of buffer in the column
and flush out the magnetically labelled cells. Repeat with other 5ml of buffer.
21- Centrifuge 1500rpm for 10 minutes, resuspend in complete medium (10% FBS) and count
CD14 +ve cells.
22- Take some cells for phenotype (FACS staining CD14/CD209/CD163/CD1a).
23- Resuspend cells at 1x106cells/ml in complete RPMI supplemented with 800U/ml GM-CSF
(0.53l/ml from the 100g/ml stock) and 500U/ml IL-4 (0.172l/ml from the 100g/ml stock).
Plate in a non-treated tissue culture dish.
Day 3:
Remove half of the medium, centrifuge and resuspend the pellet in fresh medium
supplemented with cytokines (double concentrated).
Day 5:
Remove half of the medium, centrifuge and resuspend the pellet in fresh medium
supplemented with cytokines (double concentrated).
Day 7/8:
Collect MDDCs washing the dish with PBS. Spin, resuspend in complete RPMI and count.

Phenotype: CD14/CD209/CD163/CD1a.
Immature DCs differentiated from monocytes should have lost CD14 and CD163 expression,
and have upregulated CD209 and CD1a.