Purification of cells from Purification of cells from human peripheral bloodhuman peripheral blood
• Mononuclear cells: Lymphocytes + Monocytes• Monocytes• Polymorphonuclear leukocytes (PMNs) or
granulocytes• Platelets or thrombocytes
1.060 1.070 1.080 1.090 1.100
Cell number
Density (g/ml)
MonocytesLymphocytesBasophilsNeutrophilsEosinophilsErythrocytes
Mononuclear Polymorphonuclearleukocytes
Density of human blood cells
Isolation of human mononuclear cells IDensity barrier method
MC
PMNsRBC
600g
15 min
1.077 g/ml
Axis-Shield Density Gradient Mediafor isolation of human PBMCs
• Lymphoprep™: 9.1% diatrizoate, 5.7% polysaccharide; 1.077 g/ml, 295mOsm; endotoxin <0.13 EU/ml
• Nycoprep™ 1.077: 14.1% Nycodenz®, 0.44% NaCl, 5 mM Tricine-NaOH, pH 7.0; 1.077 g/ml, 295 mOsm; endotoxin < 0.13 EU/ml
• Optiprep™ (endotoxin < 0.13 EU/ml) diluted with any suitable balanced salt solution or culture medium to give a 1.077 g/ml medium (C03)
Separation of PBMCs on Lymphoprep™
Blood:saline 1:1Blood < 12 h old
6 ml diluted blood on3 ml of Lymphoprep™
Lymphoprep™ competitors
• Ficoll-Paque (GE Health Care (ex Amersham and Pharmacia)
• Histopaque 1077 (Sigma)• Not endotoxin tested• More expensive endotoxin-tested
versions, e.g. Sigma’s Histopaque 1077 Hybrimax
Lymphoprep™ queries (I)• Anticoagulant?• Not anticoagulant sensitive• Do I have to dilute the blood?• Whole blood less easy to layer; poorer % yields• Can increase sample:Lymphoprep™ ratio• Yes – but not recommended• Can I use a leukocyte-rich plasma?• Yes, OK to separate PBMCs and PMNs• Centrifugation at 4°C rather than room
temperature?• Yes, but need to increase time by 5 min
Lymphoprep™ queries (II)• Ficoll-Paque or Histopaque work better• Impossible – media have identical composition• Poor definition of PBMC band at interface• Poor layering technique and/or brake problem• Poor separation from erythrocytes/PMNs• Blood > 12 h old; clinical specimens• Can I use it for non-human blood?• Maybe primates and some ruminants• How can I remove the platelets?
Lymphoprep™ Tube
Blood diluted1:1 with saline
800g for 15 minPBMCs
MediumdisplacedupwardsLymphoprepTM
Plastic frit
Isolation of PBMCs by flotation - mixer method (C04)
1500g
30 min
saline
1.25 mlOptiPrep
10 ml blood
PBMCs
PMNs +RBCs
Plasma +Platelets
Problem with mixer method
• Ratio of cells:plasma (haematocrit value)• Normal males: 42-54%• Normal females: 38-46%• Some clinical samples may have
significantly lower values
Removal (isolation) of platelets on a density barrier (C12)
350g
15 min
Platelets
Leukocytes+ RBCs
5 ml of 1.063 g/ml
5 ml wholeblood
Blood +OptiPrep1.095 g/ml
1.077 g/ml
saline
600g
20 min
Isolation of platelet-free PBMCs (C05)
PBMCs
PMNsPlasma + RBCs+ Platelets
• Polymorphprep™• 13.8% diatrizoate, 8.0% dextran 500• Density = 1.113 g/ml• Osmolality = 460 mOsm
Axis-Shield Density Gradient Mediafor the isolation of human PMNs from
whole blood
450-500g
30-35 min at 20oC
Whole blood5 ml
Polymorphprep5 ml
PBMCsPMNs
RBCs
Separation of PBMCs and PMNs on Polymorphprep™
321
How does Polymorphprep™ work?4
Polymorphprep™ Separation
Coulter STKR Analysis of PBMCs and Polymorphs
4 8 12 16 20 24 28 32Cell vol (femtolitres x 0.1)
Rel
ativ
e nu
mbe
r
Polymorphprep – critical points• Must use fresh (<2 h old) whole blood• Anticoagulant must be EDTA (dipotassium)• Temperature must be 18-22C• Blood:medium volume ratio must be 1:1• 5 ml + 5 ml in a 15 ml tube• Brake must NOT be used• Centrifugation time can be + 5 min• Recommended g-force is the gavav
Geometry of rotors
axis of rotationrmax rav rmin
Set rpm speed to give 500g at rmax
Equivalent to 400g at rav
Polymorphprep/sample volumes
Human PMNs from a leukocyte-rich plasma (C011)
PBMCs
PMNs
600g
20 min
1.077 g/ml
1.10 g/ml
Axis-Shield Density Gradient Mediafor the isolation of human monocytes:
barrier sedimentation (C55)
• 13.0% Nycodenz, 0.58% (w/v) NaCl, 5 mM Tricine-NaOH, pH 7.0
• Density = 1.068 g/ml• Osmolality = 335 mOsm
1.060 1.070 1.080 1.090 1.100
Cell number
Density (g/ml)
MonocytesLymphocytesBasophilsNeutrophilsEosinophilsErythrocytes
Density of human blood cells
Separation of monocytes on 335 mOsm, 1.068 g/ml barrier
1.068 g/ml
LRP600g
15 minM
L
Harvestzone
Nycoprep™ 1.068 requirements
• Leukocyte-rich plasma; not whole blood• Anticoagulant: EDTA• Blood from normal individual, < 2 h old• Room temperature 18-22°C• No brake to decelerate rotor• Centrifugation conditions may need
optimizing from lab to lab
Flotation of monocytes from leukocyte-rich plasma (C09)
600-800g
15-20 min
HBS
1.068 g/ml
1.084 g/ml
LRPLC
M
L
P
FACS analysis of monocyte band Graziani-Bowering, G.M. et al (1997)J. Immunol. Meth.,
207, 429-436
Surface Marker % eventsCD3+ 3.4
CD14+/CD4– 1.6CD14–/CD4+ 6.9CD14+/CD4+ 84.1
All monocyte markers 92.6
Monocyte flotation isolation queries
• No monocyte band observed• Band may be quite diffuse• Poor recovery and purity of monocytes• Rapid preparation of leukocytes essential
and handling of cells must be very gentle• Does method work at 4°C?• Probably
• Dilute buffered saline with water (2.5:0.5) Solution is 242 mOsm
• Dilute OptiPrep with the 242 mOsm solution (2.7: 9.3)
• Solution of 1.077 g/ml and 265 mOsm• Competition (Histopaque 1.083): 1.083
g/ml, 295 mOsm
Axis-Shield Density Gradient Media:isolation of rodent PBMCs,
sedimentation on to a barrier (C43)
1.060 1.070 1.080 1.090 1.100
Cell number
Density (g/ml)
MonocytesLymphocytesBasophilsNeutrophilsEosinophilsErythrocytes
Axis-Shield Density Gradient Mediafor the isolation of rodent PBMCs
Isotonic ammonium chloride
Rodent PMNs from leukocyte-rich plasma (C45)
MC
RBC + PMNs
600g20 min
Aspirate
1.077 g/ml265 mOsm
LRP
Common flotation strategy for purifying low density cells from
tissues
• Isolation of dendritic cells from spleen, thymus, lymph nodes etc (C20)
• Tissue disaggregated using enzymes• Cell suspension adjusted to 1.085 g/ml• Make up solution of 1.065 g/ml
Dendriticcells
600g15 min
1.065 g/ml
1.085 g/mlSample
Saline or culture
medium
Dendritic cells – flotation strategyReudl, C. et al (1996) Eur. J. Immunol., 26, 1801-1806
Pancreatic islets – flotation (C18) Van der Burg, P.P.M. (1998) Transpl. Proc. 30, 362-363
500g/5 min
Islets
Acinarcells
1.09 g/ml
1.10 g/ml
OptiPrep H’paque
Yield 92% 58%
Purity 86% 61%
Viab’lty 75% 54%
Removal of non-viable cells (C13)
800g20-25 min
Cell suspensionadjusted to1.15-1.16 g/ml
1.12 g/ml
Culture medium Viablecells
Other Axis-Shield Cell Applications
• Epithelial cells from gastric mucosa (C28)• Neurons: spinal cord (C22) brain (C29)• Human erythrocytes and reticulocytes (C35)• Megakaryocytic progenitor cells (C23/C48)• Pneumocytes and other lung cells (C25/C44)• Stellate cells from liver and pancreas (C33)• Hepatic Kupffer cells (C50)• Renal cells (C42)
Publications database on cellsPublications database on cells• OptiPrep (since 1994) over 650• Nycodenz® (since 1984) approx 2000• Using either the Applications CD or the website:• www.axis-shield-density-gradient-media.com• Follow the instructions to access the relevant
Index• Click on the cell of interest