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Purification of cells from human peripheral blood. Mononuclear cells: Lymphocytes + Monocytes Monocytes Polymorphonuclear leukocytes (PMNs) or granulocytes Platelets or thrombocytes. Monocytes. Cell number. Lymphocytes. Basophils. Neutrophils. Eosinophils. Erythrocytes. Density (g/ml).

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Purification of cells from human peripheral blood

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Purification of cells from human peripheral blood l.jpg

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 l.jpg

Monocytes

Cell number

Lymphocytes

Basophils

Neutrophils

Eosinophils

Erythrocytes

Density (g/ml)

1.060

1.070

1.080

1.090

1.100

Density of human blood cells

Polymorphonuclear

leukocytes

Mononuclear


Slide3 l.jpg

600g

15 min

MC

1.077 g/ml

RBC

PMNs

Isolation of human mononuclear cells I

Density barrier method


Axis shield density gradient media for isolation of human pbmcs l.jpg

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)


Slide5 l.jpg

Separation of PBMCs on Lymphoprep™

6 ml diluted blood on

3 ml of Lymphoprep™

Blood:saline 1:1

Blood < 12 h old


Lymphoprep c ompetitors l.jpg

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 l.jpg

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 l.jpg

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 l.jpg

Blood diluted

1:1 with saline

800g for 15 min

PBMCs

Medium

Plastic frit

displaced

LymphoprepTM

upwards

Lymphoprep™ Tube


Isolation of pbmcs by flotation mixer method c04 l.jpg

10 ml blood

saline

1500g

PBMCs

30 min

Plasma +

Platelets

PMNs +

RBCs

1.25 ml

OptiPrep

Isolation of PBMCs by flotation - mixer method (C04)


Problem with mixer method l.jpg

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 l.jpg

5 ml whole

blood

350g

15 min

Platelets

5 ml of

1.063 g/ml

Leukocytes

+ RBCs

Removal (isolation) of platelets on a density barrier (C12)


Isolation of platelet free pbmcs c05 l.jpg

saline

PBMCs

600g

1.077 g/ml

20 min

PMNs

Blood +

OptiPrep

1.095 g/ml

Plasma + RBCs

+ Platelets

Isolation of platelet-free PBMCs (C05)


Axis shield density gradient media for the isolation of human pmns from whole blood l.jpg

Axis-Shield Density Gradient Mediafor the isolation of human PMNs from whole blood

  • Polymorphprep™

  • 13.8% diatrizoate, 8.0% dextran 500

  • Density = 1.113 g/ml

  • Osmolality = 460 mOsm


Separation of pbmcs and pmns on polymorphprep l.jpg

Whole blood

5 ml

450-500g

PBMCs

30-35 min at 20oC

PMNs

5 ml

Polymorphprep

RBCs

Separation of PBMCs and PMNs on Polymorphprep™


How does polymorphprep work l.jpg

1

2

3

4

How does Polymorphprep™ work?


Polymorphprep separation l.jpg

Polymorphprep™ Separation


Coulter stkr analysis of pbmcs and polymorphs l.jpg

Relative number

4

8

12

16

20

24

28

32

Cell vol (femtolitres x 0.1)

Coulter STKR Analysis ofPBMCsandPolymorphs


Polymorphprep critical points l.jpg

Polymorphprep – critical points

  • 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


Slide21 l.jpg

axis of rotation

rmax

rav

rmin

Geometry of rotors

Set rpm speed to give 500g at rmax

Equivalent to 400g at rav


Polymorphprep sample volumes l.jpg

Polymorphprep/sample volumes


Human pmns from a leukocyte rich plasma c011 l.jpg

600g

20 min

1.077 g/ml

1.10 g/ml

Human PMNs from a leukocyte-rich plasma (C011)

PBMCs

PMNs


Axis shield density gradient media for the isolation of human monocytes barrier sedimentation c55 l.jpg

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


Density of human blood cells25 l.jpg

Monocytes

Cell number

Lymphocytes

Basophils

Neutrophils

Eosinophils

Erythrocytes

Density (g/ml)

1.060

1.070

1.080

1.090

1.100

Density of human blood cells


Separation of monocytes on 335 mosm 1 068 g ml barrier l.jpg

LRP

600g

15 min

Harvest

zone

M

L

1.068 g/ml

Separation of monocytes on 335 mOsm, 1.068 g/ml barrier


Nycoprep 1 068 requirements l.jpg

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 l.jpg

HBS

M

600-800g

1.068 g/ml

15-20 min

L

1.084 g/ml

P

LC

LRP

Flotation of monocytes from leukocyte-rich plasma (C09)


Facs analysis of monocyte band graziani bowering g m et al 1997 j immunol meth 207 429 436 l.jpg

FACS analysis of monocyte bandGraziani-Bowering, G.M. et al (1997)J. Immunol. Meth., 207, 429-436


Monocyte flotation isolation queries l.jpg

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


Slide31 l.jpg

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 l.jpg

Monocytes

Cell number

Lymphocytes

Basophils

Neutrophils

Eosinophils

Erythrocytes

Density (g/ml)

1.060

1.070

1.080

1.090

1.100

Axis-Shield Density Gradient Mediafor the isolation of rodent PBMCs


Slide33 l.jpg

Isotonic ammonium chloride

Aspirate

600g

20 min

LRP

MC

1.077 g/ml

265 mOsm

RBC + PMNs

Rodent PMNs from leukocyte-rich plasma (C45)


Common flotation strategy for purifying low density cells from tissues l.jpg

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 l.jpg

Saline or culture

medium

Dendritic

cells

600g

1.065 g/ml

15 min

Sample

1.085 g/ml

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 l.jpg

Islets

1.09 g/ml

500g/5 min

1.10 g/ml

Acinar

cells

Pancreatic islets – flotation (C18) Van der Burg, P.P.M. (1998) Transpl. Proc. 30, 362-363


Removal of non viable cells c13 l.jpg

Viable

cells

Culture medium

1.12 g/ml

800g

20-25 min

Cell suspension

adjusted to

1.15-1.16 g/ml

Removal of non-viable cells (C13)


Other axis shield cell applications l.jpg

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 cells l.jpg

Publications 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


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