TITLE: MDM culture
AUTHORS: Arpine Sokratian, enquan.xu

[DESCRIPTION]
This protocol details the steps for isolation of PBMC from donors’ buffy coat and differentiation protocol into MDM cells. We use this MDM model to investigate the immune cellular uptake and proinflammatory response upon treatment with alpha-synuclein fibrils.

[STEPS]
1. Use the buffy coat transported from Red blood cross center (on ice,  ).

2. Dilute blood with an equal amount of   with 2% Fetal Bovine Serum (PBS + 2%   )

3. Transfer the blood on top of Lymphoprep™    in the 50-ml   

4. Centrifuge at 1200 x g for 20 min Room temperature (15 - 25°C), with the brake on. If the blood has been stored for more than 2 hours, increase the centrifugation time to 30 min. 

5. Pour off the top layer, which contains the enriched MNCs, into a new tube. Do not hold the SepMate™ tube in the inverted position for longer than 2 seconds. 
6. Wash enriched PBMCs with PBS + 2% FBS. Repeat wash with 400 x gfor 10 minutes at room temperature.

7. Prepare the PBMCs at the 5 x 10^7 cells/ml within the 1 mL of PBS + 2% FBS buffer.

8. Add the sample to the 5 mL   
9. Add enrichment Cocktail to sample at 50 ul/ml from the EasySep™ Human Monocyte Enrichment Kit without CD16 Depletion   (Cat: 19058C.2). Pipet up and down for at least 5 times. Make sure you see no clump in bare eyes. It must be mixed very thoroughly, or your yield will below.

10. Incubate on icefor 10 min

11. Add 50 µL mag beads buffer (from STEMCELL TECH KIT, make sure you VORTEX the mag beads buffer 30 secs before taking50 µL out!!!) and pipet up and down for no more than 5 times (enough to see the beads mixed all well (determined by the color).

12. Incubate the mix in the refrigerator or on the ice for 5 min

13. Transfer the mix into either sterile flow tube or 15 ml canonical tube (depend on which mag selection set you would use. If you want to use the single mag set, then you have to use sterile flow tube. The multiple
channel mag set would allow you to choose between flow tube or 15 ml canonical tube. 
14. Add the selection buffer up to 2.5 ml (For flow tubes, add 2.5 mL PBS or up to 2.5 mL marker on the tube). 
15. Hold the tube (if you use the multiple channel mag set) and pour the mix buffer out into the new collection tube (15 ml canonical tube) in a continuous manner (don’t let the mix buffer back flow to contact the mag
beads trapped by the mag set on the tube wall). Don’t try to drip out the last drop of the buffer. You would rather wash twice instead of shaking of the beads to contaminate your final yield.
16. Add another 2.5 ml of selection buffer and repeat the steps listed above. Take 10 ul to mix with 90 ul of typan blue in PBS for cell counting (formula here: Total cells you count in four chamber/4x10x5x104 =total cells number in 5 ml buffer).
17. Centrifuge at 400 x g, 15 min, Room temperature, with full break and remove the selection buffer supernatant thoroughly (to remove EDTA) and resuspend with DMEM+10%FBS+1XGlutmax+ 1X Antianti+20 ng/ml human recombinant MCSF (1000x; stock should stored at-80 C). All the cytokines are from PeproTech and should be dissolved in stock 1000x except CCL2 and IL34 (100x).

18. Seed the cells into the cell culture plates (2.5 X 105/well for 24-well plate with 1 ml of cell culture medium 
19. Incubate the cell culture in CO2 cell incubator. At day 3 add 0.5 mL additional medium containing MCSF to cell culture.
MDM should be ready to use at day 7 (the cell maturation different from patient to patient, 7 days will be a safe place to use. For most of patients, if the MDM cells still don’t grow well up to 10 days, it won’t.) 

Don’t let the MDM grow more than 12 days (or the media will become yellow)