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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
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

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

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
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

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
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
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
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 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

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
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

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

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
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

Whole blood

5 ml

450-500g

PBMCs

30-35 min at 20oC

PMNs

5 ml

Polymorphprep

RBCs

Separation of PBMCs and PMNs on Polymorphprep™
coulter stkr analysis of pbmcs and polymorphs

Relative number

4

8

12

16

20

24

28

32

Cell vol (femtolitres x 0.1)

Coulter STKR Analysis ofPBMCsandPolymorphs
polymorphprep critical points
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

axis of rotation

rmax

rav

rmin

Geometry of rotors

Set rpm speed to give 500g at rmax

Equivalent to 400g at rav

human pmns from a leukocyte rich plasma c011

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
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

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
nycoprep 1 068 requirements
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

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)
monocyte flotation isolation queries
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

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

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

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
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

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

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

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
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
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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