Sample preparation for flow

cytometry
Flow cytometry webinar series
MACS Academy
Agenda

1. General introduction to sample preparation

2. Blood preparation

3. Tissue preparation
> Mechanical disruption
> Enzymatic digestion

4. Examples for tissue dissociation

> Tumor dissociation
> Dissociation of neonatal heart tissue

5. Sample clearance
> Dead cell removal
> Myelin removal
> Dead cell and doublet identification and exclusion
 Workflow of flow cytometry experiments

Sample preparation Sample labeling Acquisition Analysis

Sample preparation

Requirement for Flow Cytometry:

Single cell suspension

Viability Yield
Why is sample preparation so important?

Garbage in…

…garbage out
Factors influencing sample preparation

• Sampling methods
• Drawing blood / anti-coagulants
• Surgery
• Freezing / Thawing
• Cell culture harvest (suspension / adherent)

• Sample logistics and storage

• Duration
• Temperature
• Storage solution

• Preparation protocol
• Buffers / Reagents
• Enzymes
• Mechanical dispersion
Agenda

1. General introduction to sample preparation

2. Blood preparation

3. Tissue preparation
> Mechanical disruption
> Enzymatic digestion

4. Examples for tissue dissociation

> Tumor dissociation
> Dissociation of neonatal heart tissue

5. Sample clearance
> Dead cell removal
> Myelin removal
> Dead cell and doublet identification and exclusion
Handling of blood

• Handling of blood
• Storage of whole blood at room temperature
• Avoid mechanical stress (no shaking); cautious resuspension
• Storage of prepared cells at 4°C

• Preparation

Ficoll-Paque is a trademark of GE Healthcare companies.

• Density gradient (FicollTM) -> mononuclear cells (MNC)
> (Removal of erythrocytes, platelets and granulocytes)
• Hypotonic lysis -> MNC + granulocytes
• Whole blood (No lyse analysis, using CD45 staining)

Plasma
Diluted blood MNC
Ficoll®
Granulocytes
Ficoll
Erythrocytes
Preparation of blood

Granularity

Ficoll

Fresh blood Old blood (> 24 h)

Lysis

Good lysis Bad lysis Very bad / no lysis

Size
Agenda

1. General introduction to sample preparation

2. Blood preparation

3. Tissue preparation
> Mechanical disruption
> Enzymatic digestion

4. Examples for tissue dissociation

> Tumor dissociation
> Dissociation of neonatal heart tissue

5. Sample clearance
> Dead cell removal
> Myelin removal
> Dead cell and doublet identification and exclusion
Dissociation of tissues and organs

Tissue

Enzyme Enzyme

DNase

Mechanic Mechanic Mechanic

manual automated manual automated manual automated

 Enzymes for tissue dissociation

Enzyme Type Source Target

Trypsin serine protease bovine/porcine predominantly at c-terminal relatively harsh
pancreas side of AA Lys or Arg
Papain cysteine protease Carica papaya cleaves basic AAs relatively harsh, but less
digestive than Trypsin

Collagenase protease, includes Clostridium peptide bonds in collagen; different collagenase types
also phospholipases histolyticum Different types available, with different levels of
or neuraminidases depending on supplier unspecific activities
Roche: A, B, D, H, P
Sigma-Aldrich: I-IV,
Worthington: Type 1-4
Liberase blend of purified Clostridium histolyticum Supplied by Roche, developed for pancreatic
Blendzymes collagenases with and Bacillus polymyxa more defined activity and islet
neutral proteases or composition transplantation research
B. thermoproteolyticus
Dispase protease Bacillus polymyxa fibronectin, collagen IV separation of dermis
(collagen I) and epidermis

DNase nuclease bovine pancreas DNA avoid cell clumping

DNase I: neutral
DNase II; functions
optimally at acidic pH
MACS® Tissue Dissociation Kits

Ready-to-use, pre-titrated enzyme solutions

• Produced for Miltenyi Biotec, highly purified enzymes
• Epitope conservation
• No batch to batch variation
• Reproducible results

Pre-evaluated protocols
• Time-saving – no need for protocol development

Compatible with gentleMACS™ Dissociators

• No need for labor-intensive manual dissociation methods
• Higher reproducibility due to standardization
gentleMACS™ technology

A perfect match: innovative instruments and optimized kits

gentleMACS™ Octo Dissociator & gentleMACS™ Dissociator

Unique Dissociation Tubes:

gentleMACS™ M and C Tubes Tissue Dissociation Kits
Examples tissue preparation

Spleen Lung

Lung Dissociation
Kit, mouse

Alternative
enzyme
Agenda

1. General introduction to sample preparation

2. Blood preparation

3. Tissue preparation
> Mechanical disruption
> Enzymatic digestion

4. Examples for tissue dissociation

> Tumor dissociation
> Dissociation of neonatal heart tissue

5. Sample clearance
> Dead cell removal
> Myelin removal
> Dead cell and doublet identification and exclusion
Human breast cancer

Fat

Blood vessel

Tumor tissue
Tumor Dissociation Kit, human

RBC

Tumor Cells
GlyA-PE

TIL

CD45-FITC
Neonatal Heart Dissociation Kit, mouse and rat

Very fast protocol

Heart cell-frequencies after dissociation
Beating cardiomyocytes after dissociation
Preparation protocols on Miltenyi website

miltenyibiotec.com Web code: caad

►MACS® Sample preparation ►Applications and protocols
Agenda

1. General introduction to sample preparation

2. Blood preparation

3. Tissue preparation
> Mechanical disruption
> Enzymatic digestion

4. Examples for tissue dissociation

> Tumor dissociation
> Dissociation of neonatal heart tissue

5. Sample clearance
> Dead cell removal
> Myelin removal
> Dead cell and doublet identification and exclusion
Removal of dead cells

1. Density gradient
• Cell loss 30-50%
• Long centrifugation times and subsequent washing -> time consuming

2. Immunomagnetic depletion using Dead Cell Removal Kit

• Minimal cell loss
• Fast protocol
• Highly viable cell sample
• DNAse digestion can improve yield!
Propidium Iodide

Forward Scatter
Exclusion of dead cells during analysis

Staining with Propidium Iodide (PI) or other dead cell stains:

• Identifies dead cells
• Allows exclusion of dead cells by gating

B3

B2
Sample clearance – Myelin removal in neural samples

• For neural cell preparations from brain

• Myelin content increases with age (in mice after P10)
• Issues:
• Creates large debris fraction in prepared neural tissue
• Hampers cell analysis and isolation

• Solutions:
• Sucrose gradient; loss of large numbers of cells
• Immunomagnetic depletion (Myelin Removal Beads II)
> Efficient depletion; highly pure cells
> Minimal cell loss
Myelin removal in P22 mouse brain

3 markers proving this is the myelin

Myelin debris Cells

Exclusion of doublets during data analysis

• Aggregation of two (doublets) or more (triplets etc…) cells

• Reasons:
• Old sample
• Inappropriate storage of sample (e.g. too warm , wrong medium, too long…)
• Keeping cells in pellet after centrifugation

• Issues:
• Can form additional false positive populations
• Misinterpretation of data
Doublet identification and exclusion

SSC-Area
FSC-Height
SSC-Area

Doublets

FSC-Area
FSC-Area Singlets

FSC-
Area
Discriminating non-single cells

• Human iPS cell culture on mouse embryonic fibroblasts (mEF)

• Doublets generate false positive results / lead to wrong conclusions
Discriminating non-single cells

• Human iPS cell culture on mouse embryonic fibroblasts (mEF)

• Doublets generate false positive results / lead to wrong conclusions
Discriminating non-single cells

• Human iPS cell culture on mouse embryonic fibroblasts (mEF)

• Doublets generate false positive results / lead to wrong conlusions
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Thank you!