GWAS PBMC Isolation

 

Supplies for Blood Collection:

 

18g Needle

10ml Syringe

Purple top EDTA Tubes - 02687107

Optiprep – catalog # D1556-250ml

Molecular Grade dH20 – BP561-1 1L

15ml Falcon Tubes

2ml Eppendorf tubes C-2171

2ml Cryotubes – flat tops are easier to label

Fetal Bovine Serum (FBS)- 507533001

Cryostor CS10 – 210347

Mr. Frosty – 5100-001

Tricine – 39468100GM

NaCL- B358212

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Solutions to Prepare:

• Diluted Saline to prepare Barrier solution (Solution B)

• Barrier Solution

 

Protocol: 

• Make up the density barrier:

• Prepare Solution B: Dilute Saline (0.85g NaCL in 50ml H2O + 10ml Tricine stock solution (100mM) made fresh) with water (volume ratio 2.5:0.5 respectively); this solution has an osmolality of approx 242 mOsm. (See barrier solution calculation sheet for easy reference and calculation of quantity of barrier solution needed for number of rats. (Important** Always Keep at Room Temp)

• Prepare Barrier Solution: Dilute OptiPrep with Solution B using a volume ratio 2.8: 9.2 respectively (See barrier solution calculation sheet for easy reference and calculation of quantity of barrier solution needed for number of rats. (Important** Always Keep at Room Temp)

• Collect the 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). 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. (KEEP BLOOD AT RT)

• 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.

• In a cell culture hood - carefully layer 4 ml of diluted blood over 2 ml of the density barrier (SUPER IMPORTANT-avoid mixing at the interface –SUPER IMPORTANT) in a 15ml Falcon tube.

• Centrifuge at 700 g for 30 min at approx. 22°C in a swinging-bucket rotor with slowest acceleration and no brake (Do not use 4C it will not separate). The solution will separate into four distinct layers.

• After centrifugation the PBMCs form a sharp band at the interface.

• Gently insert the pipet tip and extract the PBMC layer. ~100-200ul depending on how sticky they are to the lower layer. Dilute by 2X in Solution B + 2% FBS and pellet the cells at 400g for 10 min to wash (X2).

• Resuspend the PBMC pellet in Cytostor10 (1000ul) and take note of pellet size 0-3 – aliquot into two tubes at 500ul each. 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.