TITLE: PBMC Isolation 
AUTHORS: Sierra Simpson, Olivier George

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Isolation of peripheral blood monocytes (PBMC) for later expansion and differentiation </div></div>

[STEPS]
?. [Solution Preparation ]
Prepare  Tricine Stock ( Fresh )    0.895g Tricine    50ml Water
?. [Blood Collection]
Collect blood by cardiac puncture into a 10ml syringe with 18g needle by slowly allowing blood to enter the syringe. Remove needle and drop blood into a purple top EDTA Tube (no vacuum – this increases hemolysis). Hemolysis can increase the "stickiness" of the PBMCs, avoid this and clotting for the cleanest preparations. 4ml collections into a single tube are most effective – the ratio of EDTA to blood is important – smaller volumes of blood do not separate as well and yield far less cells.
?. [Solution Preparation ]
Prepare Buffered Saline Solution (Solution B) Dissolve 0.85g of NaCl in 50 ml waterAdd 10 ml of Tricine stock solutionAdjust to pH 7.0 with 1 M NaOH   Volumize to 100 ml. 

Adjust to pH 7.0 with 1 M NaOH
0 Room temperature
?. [Solution Preparation ]
Prepare Density Barrier Dilute Solution B with molecular grade water (volume ratio 2.5:0.5 respectively) Check osmolality of the solution - should be 242 +/- 10Dilute OptiPrepTM with this solution using a volume ratio 2.7: 9.3 respectively Sterile filter in a hood before use* See Barrier Solution calculation sheet to determine the quantity needed for the number of animals being harvested
0 Room temperature
?. [Solution Preparation ]
Prepare Solution B + 2% Fetal Bovine Serum Mix 50ml Solution B + 1ml  FBSSterile filter in a hood before use
0 Room temperature
?. [PBMC Isolation ]
In a cell culture hood - dilute the blood (2ml) with an equal volume of Solution B + 2% FBS. Save 2x500ul aliquots of FBS used as a control for later experiments. FBS is essential to allow PBMC to not stick to lower layer of density gradient, otherwise cells are much harder to isolate and there are significant numbers of RBCs that are difficult to wash out.
?. [PBMC Isolation ]
In a cell culture hood - carefully layer 4 ml of diluted blood over 2 ml of the Density barrier in a 15ml Falcon TubeMaintain a 2:1 ratio of blood to density barrier if there is more blood available  - eg. 6ml diluted blood to 3ml barrier(SUPER IMPORTANT- avoid mixing at the interface –SUPER IMPORTANT) This requires a slow and steady hand to carefully pipet the blood over the density barrier. Take your time!
?. [PBMC Isolation ]
Centrifuge at 700 g for 30 min at   in a swinging-bucket rotor with slowest acceleration and deceleration with no brake. Do not use 4C it will not separateDo not use the brake as it will disrupt the formation of the layers.  It will take ~10 min for the rotor to stop spinning after the centrifugation step as there is no brake.
0 Room temperature
?. [PBMC Isolation ]
After centrifugation the PBMCs form a sharp band at the interface.
?. [PBMC Isolation ]
Gently insert the pipet tip and extract the PBMC layer. ~100-200ul depending on how sticky they are to the lower layer. Sometimes there is really nice separation, but sometimes the PBMCs are stuck directly to the blood layer below. Avoid picking up the erythrocytes if possible.
?. [PBMC Isolation ]
Dilute by 2X in Solution B + 2% FBS and pellet the cells at 400g for 10 min to wash (X2).
?. [PBMC Isolation ]
Resuspend the PBMC pellet in Cytostor10 (250ul) and take note of pellet size 0-3 Transfer aliquot to a white top cryotube with appropriate labeling
?. [PBMC Isolation ]
Place resuspended cells in Mr. Frosty o/n to reach  -80 for at least 24h, at which point the cells can be transferred to the liquid nitrogen. Make sure the Mr. Frosty has enough isopropanol in the bottom container to reach the line labeled on the side.