[PPT]PowerPoint Presentationaxis-shield-density-gradient-media.com/training-4new.ppt · Web viewThis graphic shows the density of human blood cells. The polymorphonuclear leukocytes

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