Upload
oliver-stonehill
View
230
Download
3
Embed Size (px)
Citation preview
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
Mediumdisplacedupwards
LymphoprepTM
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
PBMCs
PMNs
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 32
Cell 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
LRP
600g
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 % 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
• 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
600g
20 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
600g
15 min1.065 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)
800g
20-25 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