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