Version 2 / 15.02.2012
Sorting Cord Blood Progenitor Populations
Sample preparation (Isolation of MNCs by density gradient centrifugation)
 Remove the cord blood from the bag under sterile conditions
 Rotate bag if there are clots
 Cord Bag usually contains 50g 50-75mls blood (Sysmex sample 250μl optional)
 Dilute 1 + 1 PBS @ RT (e.g. 50 ml + 50ml) ideally final volume = even no of x25ml
 Aliquot 12.5ml Ficoll-Paque™ per 50ml falcon
 Carefully pipette 25ml of cell suspension on the ficoll
 Spin 30mins, 1410rpm ↑3,↓0 brake off
 Carefully recover PBMC layer
 Pool 2-3 fractions per tube
 Fill to 50mls with PBS to remove more platelets
 Spin 6 mins, 1600rpm ↑9,↓0
 Remove S/N gently
 Re-suspend in total 50ml MACS buffer x 1 tube (cold MACS)
 Count cells (Sysmex sample 250μl optional)
 Spin 6mins, 1600rpm ↑9,↓0
 Remove S/N carefully
Isolation of CD34+ progenitors from cord blood using EasySep® progenitor cell enrichment
with Platelet Depletion (#19056)
 Keep buffers and cells cool to avoid antibody capping
 Re-suspend cells at 5 x 107 / ml in MACS buffer x 1
 Transfer suspension to 5ml FACS tubes (2ml per tube)
 Add Human Progenitor Cell Enrich. Cocktail with Platelet depletion at 50μl / ml
 Mix well and incubate for 15mins at RT
 Add Magnetic Nanoparticles to cell suspension at 50μl / ml (mix nanoparticle tube well before
adding)
 Mix suspension well and incubate for a further 15mins at RT
 Bring suspension up to a total volume of 2.5mls using MACS buffer x 1and mix cells by
gently pipetting
 Place tube in magnet and leave for 10mins
 Invert magnet and pour contents into a clean tube in one continuous motion
 DO NOT shake off any drops that may remain in the tube mouth
 Unwanted cells remain bound inside the original tube
 Count cells
Antibody staining for FACS
 Distribute cell suspension into control and sorting tubes accordingly (require a minimum of 5 x
104 cells for each control tube)
 Spin 6mins, 1600rpm ↑9,↓0
Page 1 of 3
Version 2 / 15.02.2012
Control tubes
Re-suspend control tubes in FACS buffer and stain as follows:
(test = a final volume of 100μl)
1. Unstained CD34-enriched
2. FITC (CD38 #BD 560982) 20μl / test
3. PE (CD90 #BD 555596) 5μl / test
4. PerCP (CD10 #Biolegend 312215) 5μl / test
5. PECy5 (CD10 #BD 555376) 20μl test
6. PECy7 (CD90 #BD 561558) 5μl / test
7. Pacific Blue (CD45RA #Invitrogen MHCD45RA28) 5μl / test
8. APC (CD34 #BD 560940) 20μl / test
Sorting tube
Re-suspend sorting tube in 80μl FACS buffer and add 20μl Fc inhibitor (#eBioscience 14-9161)
and incubate at 4oC for 20mins. Proceed to stain without washing:
9. Lin- PECy5 (CD2 #BD 555328 20μl / test; CD3 #BD 561006 20μl / test; CD11b #BD
561686 20μl / test; CD14 #Invit. MHCD1418 5μl / test; CD19 #BD 555414 20μl / test;
CD56 #BD 555517 20μl / test; GPA #NHS 1/100)
CD38 FITC (#BD 560982 20μl / test)
CD34 APC (#BD 560940 20μl / test)
CD90 PECy7 (#BD 561558 5μl / test)
CD45RA Pacific Blue (#Invitrogen MHCD45RA28 5μl / test)
CD123/IL-3Ra PE (#eBioscience 12-1239-42 5μl / test)
CD10 PerCP (#Biolegend 312215 5μl / test)
 Incubate for 20mins at 4oC
 Wash with 2ml FACS buffer
 Spin 6mins, 1600rpm ↑9,↓0
 Remove S/N gently and re-suspend in 500μl FACS buffer
This strategy will enable sorts for the following populations:
HSC: Lin-CD34+CD38-CD90+CD45RAMPP: Lin-CD34+CD38-CD90-CD45RACLP: Lin-CD34+CD38+CD10+CD45RA+
CMP: Lin-CD34+CD38+IL-3RalowCD45RAMEP: Lin-CD34+CD38+IL-3Ra-CD45RAGMP: Lin-CD34+CD38+IL-3Ra+CD45RA+
Materials
EasySep® progenitor cell enrichment kit with CD41 depletion (#19057)
PBS (Sigma, Cat. #D8537) 500ml RT
CellGro CellGenix Cat # 2001 SCGM 500ml
VWR 5ml Round-bottom tubes
MACs buffer
PBS (Sigma, Cat#D8537) 500ml
0.5M EDTA (Sigma, Cat E7889 50ml) 2ml (2mM final)
BSA (Sigma, Cat#A9576) 30% 8.33ml (0.5% final)
Store 4°C
Page 2 of 3
Version 2 / 15.02.2012
FACS buffer
PBS (Sigma, Cat#D8537) 500ml
0.5M EDTA (Sigma, Cat E7889 50ml) 5ml (5mM final)
BSA (Sigma, Cat#A9576) 30% 3.3ml (0.2% final)
Store 4°C
NHS antibodies available at http://ibgrl.blood.co.uk
International Blood Group Reference Laboratory
NHSBT Filton
500 North Bristol Park
Northway
Filton
Bristol BS34 7QH
UK
Page 3 of 3