Transcript Document

Purification of cells from
human peripheral blood
• Mononuclear cells: Lymphocytes + Monocytes
• Monocytes
• Polymorphonuclear leukocytes (PMNs) or
granulocytes
• Platelets or thrombocytes
Density of human blood cells
Cell number
1.060
1.070
Monocytes
Lymphocytes
Basophils
Neutrophils
Eosinophils
Erythrocytes
1.080
1.090
1.100 Density (g/ml)
Mononuclear Polymorphonuclear
leukocytes
Isolation of human mononuclear cells I
Density barrier method
600g
15 min
1.077 g/ml
MC
RBC
PMNs
Axis-Shield Density Gradient Media
for 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 1:1
Blood < 12 h old
6 ml diluted blood on
3 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)
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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)
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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 diluted
1:1 with saline
800g for 15 min
PBMCs
Plastic frit
LymphoprepTM
Medium
displaced
upwards
Isolation of PBMCs by flotation mixer method (C04)
10 ml blood
saline
1.25 ml
OptiPrep
1500g
PBMCs
30 min
Plasma +
Platelets
PMNs +
RBCs
Problem with mixer method
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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)
5 ml whole
blood
5 ml of
1.063 g/ml
350g
15 min
Platelets
Leukocytes
+ RBCs
Isolation of platelet-free PBMCs (C05)
saline
PBMCs
600g
1.077 g/ml
Blood +
OptiPrep
1.095 g/ml
20 min
PMNs
Plasma + RBCs
+ Platelets
Axis-Shield Density Gradient Media
for the isolation of human PMNs from
whole blood
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Polymorphprep™
13.8% diatrizoate, 8.0% dextran 500
Density = 1.113 g/ml
Osmolality = 460 mOsm
Separation of PBMCs and PMNs
on Polymorphprep™
5 ml
Whole blood
450-500g
30-35 min at 20oC
5 ml
Polymorphprep
PBMCs
PMNs
RBCs
How does Polymorphprep™ work?
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Polymorphprep™ Separation
Relative number
Coulter STKR Analysis of PBMCs and
Polymorphs
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12
16
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28
Cell vol (femtolitres x 0.1)
32
Polymorphprep – critical points
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Must use fresh (<2 h old) whole blood
Anticoagulant must be EDTA (dipotassium)
Temperature must be 18-22C
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 gav
Geometry of rotors
rmax rav rmin axis of rotation
Set rpm speed to give 500g at rmax
Equivalent to 400g at rav
Polymorphprep/sample volumes
Human PMNs from a leukocyterich plasma (C011)
600g
20 min
PBMCs
1.077 g/ml
1.10 g/ml
PMNs
Axis-Shield Density Gradient Media
for 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
Density of human blood cells
Cell number
1.060
1.070
Monocytes
Lymphocytes
Basophils
Neutrophils
Eosinophils
Erythrocytes
1.080
1.090
1.100 Density (g/ml)
Separation of monocytes on 335
mOsm, 1.068 g/ml barrier
LRP
600g
15 min
1.068 g/ml
M
L
Harvest
zone
Nycoprep™ 1.068 requirements
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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)
HBS
M
600-800g
1.068 g/ml
15-20 min
L
1.084 g/ml
LC
LRP
P
FACS analysis of monocyte band
Graziani-Bowering, G.M. et al (1997)J. Immunol. Meth.,
207, 429-436
Surface Marker
% events
CD3+
3.4
CD14+/CD4–
1.6
CD14–/CD4+
6.9
CD14+/CD4+
84.1
All monocyte markers
92.6
Monocyte flotation isolation
queries
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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
Axis-Shield Density Gradient Media:
isolation of rodent PBMCs,
sedimentation on to a barrier (C43)
• 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
for the isolation of rodent PBMCs
Cell number
1.060
1.070
Monocytes
Lymphocytes
Basophils
Neutrophils
Eosinophils
Erythrocytes
1.080
1.090
1.100 Density (g/ml)
Rodent PMNs from leukocyte-rich
plasma (C45)
Aspirate
600g
20 min
LRP
MC
1.077 g/ml
265 mOsm
RBC + PMNs
Isotonic ammonium
chloride
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
Dendritic cells – flotation strategy
Reudl, C. et al (1996) Eur. J. Immunol., 26, 1801-1806
Saline or
culture
medium
1.065 g/ml
Sample
1.085 g/ml
Dendritic
cells
600g
15 min
Pancreatic islets – flotation (C18)
Van der Burg, P.P.M. (1998) Transpl. Proc. 30, 362-363
OptiPrep H’paque
1.09 g/ml
1.10 g/ml
Islets
Yield
92%
58%
500g/5 min
Purity
86%
61%
Viab’lty
75%
54%
Acinar
cells
Removal of non-viable cells (C13)
Culture medium
1.12 g/ml
800g
20-25 min
Cell suspension
adjusted to
1.15-1.16 g/ml
Viable
cells
Other Axis-Shield Cell Applications
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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 cells
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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