Lab 2. Methods of Leukocyte fractionation:
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Lab 2. Methods of Leukocyte fractionation: Enrichment: 1. Fluorescence activated cell sorting (FACS) -cost, -availability of equipment -markers may be on more than one cell type e.g. MHC class II on APCs (DC, M, B cells) -markers may not be on a cell type

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Lab 2. Methods of Leukocyte fractionation: Enrichment:

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Lab 2 methods of leukocyte fractionation enrichment

Lab 2. Methods of Leukocyte fractionation:

Enrichment:

1. Fluorescence activated cell sorting (FACS)

-cost,

-availability of equipment

-markers may be on more than one cell type

e.g. MHC class II on APCs (DC, M, B cells)

-markers may not be on a cell type

e.g. no Ig on plasma cells

-availability of Abs or MHC tetramers

(don’t have antibodies for cell surface markers in many species)


Lab 2 methods of leukocyte fractionation enrichment

1. FACS isolation with specific antibodies or tetramers.

Antibody identifies specific cell type (eg. CD3+ T cells)

Tetramer of MHC class I with specific peptide for isolation of specific T cells (eg. influenza specific CD8+ T cells)


Lab 2 methods of leukocyte fractionation enrichment

2. Panning or Magnetic Beads (MACS) for positive or negative selection

2.a Magnetic sorting

The magnetically labeled fraction is recovered by applying the vessel to an external magnet.

The magnetic fraction can be washed and isolated for subsequent use in downstream procedures,

or the supernatant is recovered for further use.


Lab 2 methods of leukocyte fractionation enrichment

2 b. Cell separation using an AutoMacs magnetic cell sorter

The splenocytes are washed (following removal of erythrocytes by osmotic shock) and incubated with

anti-CD43 and anti-Mac-1 antibody-conjugated microbeads (Miltenyi Biotec).

The bead-bound cells (positive fraction) are separated from unbound cells (negative fraction).


Lab 2 methods of leukocyte fractionation enrichment

3. Depletion of non-target cells-examples

-C’ mediated lysis of a non-target cell- need Abs

4. Enrichment in vivo -e.g. peritoneal priming and lavage

5. Removal of cells adherent to nylon wool.

(monocytes stick to nylon wool)


Lab 2 methods of leukocyte fractionation enrichment

6. Continuous gradient centrifugation for cell separation based

-cell fractionation by differential fractionation

-density gradient fractionation

7. Discontinuous gradient centrifugation-

-granulocytes, RBCs and other dense cells removed

-physical characteristics may not distinguish cell types

8. Cytospin to concentrate, stain and visualize cells.


Lab 2 methods of leukocyte fractionation enrichment

DENSITY

DENSITY is the mass per unit volume of a substance, often expressed in g/ml.

DIFFERENTIAL CENTRIFUGATION

DIFFERENTIAL CENTRIFUGATION separates particles on the basis of their size.

By a series of centrifugations at various speeds and times, different-sized particles are sedimented

and collected from an initially homogenous suspension.

DENSITY GRADIENT CENTRIFUGATION

DENSITY GRADIENT CENTRIFUGATION is separation performed in a supporting

column of solution in which the density and solution concentration increase toward the bottom

of the centrifuge tube.

Requires a gradient maker to set up.

DISCONTINUOUS, OR STEP, GRADIENT

DISCONTINUOUS, OR STEP, GRADIENT is one composed of layers, with abrupt changes in density

and/or concentration from one layer to the next.


Lab 2 methods of leukocyte fractionation enrichment

6. Continuous gradient centrifugation

a. Differential centrifugation for fractionation

Fractionation of a cell homogenate to isolate nuclei or smaller parts of cell.


Lab 2 methods of leukocyte fractionation enrichment

6.b. continuous gradient centrifugation

-Density gradient centrifugation

A gradient maker is used

to mix liquids of two densities

Close the port between the solutions. Add lower density solution to the output side,

higher density to the other. Need rapid mixing in the chamber and make sure you see

the higher concentration solution mixing in. Fill centrifuge tubes from the bottom with

liquid of increasingly higher density displacing the lower density liquid until the tube is full.

Usually a long metal syringe needle is placed in the bottom of the tube so that the density is

not really disturbed when the needle is removed.


Lab 2 methods of leukocyte fractionation enrichment

6. b. Continuous gradient centrifugation

low

Monocytes

Lymphocytes

PMNs

RBCs

spin

high

To wash isolated cells, resuspend in media and pellet again.

Cells are pelleted intact at low speeds 400 x g for 10 minutes


Lab 2 methods of leukocyte fractionation enrichment

6. b. Continuous gradient separation:

Separation on a preparative scale by centrifugation

through a sucrose gradient.

Two types of

hepatitis B capsid

Separation and crystallization of T = 3 and T = 4 icosahedral complexes of the

hepatitis B virus core protein. Acta Cryst. (1999). D55, 717-720


Lab 2 methods of leukocyte fractionation enrichment

7. Step Gradient-discontinuous gradient centrifugation

Layering blood on Histopaque 1077

serum

“buffy coat” or PBL

Red blood cells,

Platelets, granulocytes

Histopaque

Unfractionated Blood on Histopaque

(note the undisturbed interface)

Fractionated blood on Histopaque


Lab 2 methods of leukocyte fractionation enrichment

7.a. Separation of heparinized blood on Histopaque 1077

-one step

Lymphocytes/monocytes

Red blood cells, platelets

granulocytes.


Lab 2 methods of leukocyte fractionation enrichment

7.b. Step gradient-multiple steps

Percoll density centrifugation for fractionation of B lymphocytes

Mature B cells

Proliferating B cells

Plasma cells

Trout cells were layered over step gradients of 50, 60, 70% Percoll.

Western blot analysis of Pax-5 and Blimp-1 was used to test the purification. These two transcription factors display distinct and partially overlapping expression patterns during B cell differentiation.

mature (resting and early activated) B cells (Pax-5+Blimp–) reside in the 70% layer,

proliferating B cells reside in the 60% layer (Pax-5+Blimp+)

and plasma cells (Pax-5–Blimp+) reside in the 50% layer.

J Immunol. 2005 Jun 1;174(11):6608-16.


Lab 2 methods of leukocyte fractionation enrichment

8. A cytospin instrument is used to collect cells from a solution

for display on a slide.

A cytospin device


Lab 2 methods of leukocyte fractionation enrichment

Centrifugation basics

Catastrophic failure of an

unbalanced centrifuge

Always balance opposing tubes

Place test tube in centrifuge holder.

The higher the speed the more

accurately balanced the centrifuge

must be.

Never walk away from a

Balance with another test tube

filled to the same level

in the opposite holder.


Lab 2 methods of leukocyte fractionation enrichment

Centrifuge Basics-continued

Make sure you are using the correct rotor for the centrifuge.

Never exceed the maximum g force for the rotor.

Make sure you are using the correct centrifuge tube for the job.

Tubes may break if centrifuged at too high a speed.

Even plastic tubes can break if not in appropriate adaptors.

Pre-cool centrifuge and rotors.

NEVER walk away from a centrifuge until it reaches top speed.


Lab 2 methods of leukocyte fractionation enrichment

Use of a centrifuge nomogram for conversion of rpm to rcf.

Q. You are following a

protocol that says:

Spin cells at 400 rpm for

10 minutes. What else do

you need to know?

RCF=11.2 x r (RPM/1000)2

r in cm


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