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Purification of cells from human peripheral blood PowerPoint PPT Presentation


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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

  • Mononuclear cells: Lymphocytes + Monocytes

  • Monocytes

  • Polymorphonuclear leukocytes (PMNs) or granulocytes

  • Platelets or thrombocytes


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


600g

15 min

MC

1.077 g/ml

RBC

PMNs

Isolation of human mononuclear cells I

Density barrier method


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™

6 ml diluted blood on

3 ml of Lymphoprep™

Blood:saline 1:1

Blood < 12 h old


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?


Blood diluted

1:1 with saline

800g for 15 min

PBMCs

Medium

Plastic frit

displaced

LymphoprepTM

upwards

Lymphoprep™ Tube


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

  • Ratio of cells:plasma (haematocrit value)

  • Normal males: 42-54%

  • Normal females: 38-46%

  • Some clinical samples may have significantly lower values


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)


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 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


Whole blood

5 ml

450-500g

PBMCs

30-35 min at 20oC

PMNs

5 ml

Polymorphprep

RBCs

Separation of PBMCs and PMNs on Polymorphprep™


1

2

3

4

How does Polymorphprep™ work?


Polymorphprep™ Separation


Relative number

4

8

12

16

20

24

28

32

Cell vol (femtolitres x 0.1)

Coulter STKR Analysis ofPBMCsandPolymorphs


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


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


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 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


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


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

  • 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


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


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


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


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


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

  • 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


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


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


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

  • 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

  • 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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