Separating Cells 1st Edition by Dipak Patel 1859961487 9781859961483

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ISBN 10: 1859961487

ISBN 13: 9781859961483

Author: Dipak Patel

Separating Cells: The basics provides user-friendly and practical guidance to the techniques most commonly used to separate cells. The book offers a concise overview of the fundamental principles and explains the ‘what, how and why’. This title will be of considerable interest to newcomers to these techniques.

Separating Cells 1st Table of contents:

Chapter 1 Preparation of cell suspensions

1 Diversity of cells

2. Why separate living cells?

3 Preparation of suspensions of single cells

3.1 Isolation of cells from suspension

3.2 Isolation of cells from solid tissues

Animal and human tissue

Other tissue

4 Viability measurements

5 Separation of viable and nonviable cells

6 Characterization of cells

6.1 Light microscopy

6.2 Phase-contrast microscopy

6.3 Electron microscopy

6.4 Confocal microscopy

6.5 Flow cytometry

6.6 Magnetic beads

6.7 Immunohistochemistry

Cell preparation

Fixatives

Antibodies

Mounting media

Detection method

6.8 Metabolic characteristics

Further reading

References

Protocol 1.1

Equipment

Reagents

Protocol

Protocol 1.2 Isolation of cells from peritoneal lavage

Equipment

Reagents

Protocol

Notes

Protocol 1.3 Isolation of cells from soft tissue by mechanical disruption

Equipment

Reagents

Protocol

Notes

Protocol 1.4 Preparation of vascular smooth muscle cells by mechanical disruption and outgrowth in culture

Equipment

Reagents

Protocol

Notes

Protocol 1.5 Preparation of endothelial cells from omental fat by mechanical disruption and enzymatic digestion

Equipment

Reagents

Protocol

Notes

Protocol 1.6 Isolation of neurons from rat ganglia by mechanical disruption and enzymatic digestion (Lindsay et al. 1991)

Equipment

Reagents

Protocol

Notes

Protocol 1.7 Disaggregation of embryonic and normal and malignant tissues

Equipment

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Protocol

Notes

Protocol 1.8 Separation of renal proximal tubule cells by tissue swelling and enzymatic digestion

Equipment

Reagents

Protocol

Notes

Protocol 1.9 Preparation of rat hepatocytes by continuous perfusion with enzyme solution

Equipment

Reagents

Protocol

Notes

Protocol 1.10 Release of cultured cells from substratum by trypsinization

Equipment

Reagents

Protocol

Notes

Protocol 1.11 Isolation of yeast protoplasts (spheroplasts)

Equipment

Reagents

Protocol

Notes

Protocol 1.12 Isolation of plant protoplasts

Equipment

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Protocol

Notes

Protocol 1.13 Cell viability determined by trypan blue exclusion

Equipment

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Protocol

Notes

Protocol 1.14 Cell viability determined by fluorescence staining methods

Equipment

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Protocol

Notes

Protocol 1.15 Cell viability determined by protein synthesis

Equipment

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Protocol

Notes

Protocol 1.16 Cell viability determined by uptake of radiolabeled thymidine

Equipment

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Protocol

Notes

Protocol 1.17 Cell preparation by cytocentrifuge for immunostaining

Equipment

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Protocol

Notes

Protocol 1.18 Characterization of cells by indirect immunofluorescence staining

Equipment

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Protocol

Notes

Protocol 1.19 Indirect (DAB) immunoperoxidase staining

Equipment

Reagents

Protocol

Notes

Protocol 1.20 Metabolic characterization of endothelial cells

Equipment

Reagents

Protocol

Notes

Chapter 2 Fractionation of cells by sedimentation methods

1 Introduction

2 Separation media

3 Iso-osmotic gradients

3.1 Percoll gradients

3.2 Nonionic iodinated media

3.3 Ficoll gradients

3.4 Preparation of iso-osmotic gradients

Discontinuous iso-osmotic gradients

Continuous iso-osmotic gradients

4 Choice of separation method

5 Separation of cells on the basis of size

6 Separation of cells on the basis of their density

6.1 Fractionation of blood cells using density-barrier methods

Platelets

Mononuclear cells

Polymorphonuclear cells

Erythrocytes

6.2 The purification of viable spermatozoa from bovine semen

6.3 The separation of viable and nonviable cells from disaggregated tissue and lavages

6.4 The fractionation of cells from disaggregated rat liver

6.5 Isolation of protoplasts from digested plant tissue

6.6 Enhanced isopycnic separation of cells by density perturbation

7 Concluding remarks

References

Protocol 2.1 Isolation of platelets from human peripheral blood (Ford et al., 1990)

Equipment

Reagents

Protocol

Notes

Protocol 2.2 The isolation of mononuclear cells from diluted blood (Bøyum, 1968)

Equipment

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Protocol

Notes

Protocol 2.3 The isolation of mononuclear cells from whole blood (Ford and Rickwood, 1990; 1992)

Equipment

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Protocol

Note

Protocol 2.4 The fractionation of human mononuclear cells (Bøyum, 1983)

Equipment

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Protocol

Note

Protocol 2.5 The isolation of polymorphonuclear cells from blood

Equipment

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Protocol

Notes

Protocol 2.6 Isolation of erythrocytes

Equipment

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Protocol

Protocol 2.7 The isolation of viable spermatozoa from bovine semen using Nycodenz gradients

Equipment

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Protocol

Note

Protocol 2.8 The isolation of viable spermatozoa from bovine semen using OptiPrep gradients

Equipment

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Protocol

Note

Protocol 2.9 Purification and enrichment of viable cells prior to further fractionation

Equipment

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Protocol

Note

Protocol 2.10 Isopycnic separation of rat liver cells using continuous Percoll gradients (Pertoft et al., 1977)

Equipment

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Protocol

Note

Protocol 2.11 Isolation of hepatocytes by differential pelleting

Equipment

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Protocol

Note

Protocol 2.12 Fractionation of sinusoidal cells (Brouwer et al., 1992)

Equipment

Reagents

Protocol

Sample preparation

Gradient preparation and centrifugation

Analysis of gradients

Protocol 2.13 The isolation and purification of plant protoplasts using OptiPrep gradients

Equipment

Reagents

Protocol

Determination of leaf osmolality (Sarhan and Cesar, 1988)

Protoplast isolation (Sarhan and Cesar, 1988)

Centrifugation

Note

Protocol 2.14 Isolation of immunologically distinct cell subpopulations using density perturbation methods

Equipment

Reagents

Protocol

Preparation of beads for labeling cells

Bead binding by cells

Fractionation of cells

Chapter 3 Centrifugal elutriation

1 Introduction

2 Principles

3 Equipment

3.1 Centrifuge

3.2 Separation chamber and rotor assembly

3.3 Pump

4 Conditions affecting separation

5 Applications of centrifugal elutriation

5.1 Synchronized cells

5.2 Blood cells

5.3 Liver cells

6 Advantages and disadvantages

References

Protocol 3.1 Standard procedure for cell separation by centrifugal elutriation

Equipment

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Protocol

Notes

Protocol 3.2 Separation of growth synchronous cells

Equipment

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Protocol

Notes

Protocol 3.3 Purification of rat hepatocyte couplets

Equipment

Reagents

Protocol

Notes

Chapter 4 Free-flow electrophoresis

1 Introduction

2 Main types of FFE equipment

2.1 ElphorVAP

2.2 Octopus-PZE

3 Buffers

3.1 Separation chamber buffers

Low ionic media

Triethanolamine-free media

3.2 Electrode chamber buffers

4 Preparation of cell samples for FFE

4.1 Types of cells separated by FFE

4.2 Purification and preparation of cell samples before FFE

5. FFE of cell samples

5.1 Preparation of FFE apparatus

5.2 Construction of cell fractionation profiles

5.3 Evaluation of cells separated by FFE

6 Factors affecting FFE

6.1 Cell clumping

6.2 Leaks

6.3 Bacterial contamination and blockages

6.4 Temperature

6.5 pH and conductivity

6.6 Filter membranes

7 Applications of FFE

References

Protocol 4.1 Purification and preparation of lymphocytes for FFE (Hansen et al., 1989)

Equipment

Reagents

Protocol

Protocol 4.2 Purification and preparation of platelets for FFE (Crook and Crawford, 1989)

Equipment

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Protocol

Note

Protocol 4.3 Purification and preparation of neutrophils for FFE (Eggleton et al., 1989)

Equipment

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Protocol

Note

Protocol 4.4 Purification and preparation of kidney cells for FFE (Kreisberg et al., 1977)

Equipment

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Protocol

Protocol 4.5 Purification and preparation of ascitic human cells for FFE (Pretlow et al., 1981)

Equipment

Reagents

Protocol

Protocol 4.6 Re-electrophoresis of FFE separated cells

Protocol

Note

Chapter 5 Affinity separation

1 Introduction

2 Separation by nonmagnetic techniques

2.1 Affinity to fibers and solid surfaces

2.2 Affinity chromatography

3 Separation by magnetic techniques

3.1 Immunomagnetic beads

3.2 Magnetic cell sorting using MACS Microbeads

3.3 Magnetic cell sorting using Dynabeads

3.4 Type of separation

3.5 Detachment of positively selected cells

3.6 Factors affecting immunomagnetic separation

Target cells

Antibodies

Other variables

3.7 Applications

References

Protocol 5.1 Isolation of monocytes by adherence to solid surfaces

Equipment

Reagents

Protocol

Note

Protocol 5.2 Isolation of HLA-DR positive lymphocytes using antibody-antigen binding

Equipment

Reagents

Protocol

Preparation of antibody column

Isolation of HLA-DR positive lymphocytes

Note

Protocol 5.3 Direct positive selection of CD4+ T cells from whole blood

Equipment

Reagents

Protocol

Preparation of Dynabeads M-450 CD4

Isolation of cells

Notes

Protocol 5.4 Preparation of antibody-coated cells for use in indirect positive selection

Equipment

Reagents

Protocol

Notes

Chapter 6 Flow cytometry

1 Introduction

2 The flow cytometer—instrumentation

2.1 Light source

2.2 Flow chamber

2.3 Optical components

2.4 Light scatter

2.5 Signal detection and processing

2.6 Fluorescence

2.7 Gating

3 Types of flow sorting

3.1 Cell sorting by droplet formation

3.2 Cell sorting by deflection of the sample stream

4 Factors affecting cell sorting

5 Separation—purity, yield and sterility

6. Applications

Further reading

References

Protocol 6.1 Checklist for initial steps in setting up a flow sorter

Protocol

Notes

Protocol 6.2 Guide to checking droplet delay time

Procedure

Notes

Protocol 6.3 Checklist for measuring the yield of sorted cells

Protocol

Protocol 6.4 Precautions taken to maintain sterility

Procedure

Sheath tubing

Sample collection

Sample delivery

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