0 103 104 105

CD45RA FITC

0

-10 2

102

103

104

105

PE0 103 104 105

CD45RA FITC

0

-10 2

102

103

104

105

Com

p-PE

-A ::

PE-

A

T cells60.4

0 103 104 105

CD3

0

500

1.0K

1.5K

2.0K

Cou

nt

Forward and side scatter may not remove all dead cells

bio-rad.com/zoeDoublets can give false positives

A viability dye is a better way to exclude dead cells and improve your databio-rad-antibodies.com/viability

0 50K 100K 150K 200K 250K

FSC-Area

0

50K

100K

150K

200K

250K

FSC

-Hei

ght

live87.6

0 103 104 105

Propidium Iodide

0

50K

100K

150K

200K

250K

SSC

0 103 104 105

CD3

0

-10 2

102

103

104

105

CD

14

Cold alcohol � xation for nuclear staining

intact cells35.1

0 50K 100K 150K 200K 250K

FSC

0

50K

100K

150K

200K

250K

SSC

0 103 104 105

CD3

0

-10 2

102

103

104

105

CD

14

87.6

400 500 600 700

Wavelength, nm800

Be P

repar

ed

Help

ful T

ips

Understand the biology

Remove the dead

Remove the doublets

Consider controls Know your cytometer

Know the lineage expression, antigen density, cell frequency and size.bio-rad-antibodies.com/markers

Perform the right controls for your experiment.bio-rad-antibodies.com/fc-controls

Prepare your samples carefully, in cold buffer, at the right concentration and remove clumps.bio-rad-antibodies.com/cellprep

Select your marker, choose your antibody, � nd compatible � uorophores and build your panel.bio-rad-antibodies.com/fl uorophores

Understand your cytometer.bio-rad-antibodies.com/ZE5bio-rad-antibodies.com/S3e

Select your antibody and fl uorophore

Prepare your sample

To view our full range of products optimized for � ow cytometry visit bio-rad-antibodies.com/� owBio-Rad

Laboratories, Inc.

bio-rad.com/zoebio-rad.com/zoe

Granulocytes10.9

Lymphocytes11.5

Monocytes3.10

0 50K 100K 150K 200K 250K

FSC-A

0

50K

100K

150K

200K

250K

SSC

-A T cells63.4

0 103 104 105

CD3 A647

0

200

400

600

800

Cou

nt

0 50K 100K 150K 200K 250K

PI

0

200

400

600

800

Cou

nt

Q161.8

Q20.83

Q334.1

Q43.25

0 103 104 105

CD8

0

-10 2

102

103

104

105

CD

4

Surface staining

Optional red cell lysisErythrolyse (BUF04A)

Intracellularstaining

Nuclearstaining

Flow

Pro

toco

ls

Permeabilize and � x for intracellular staining using Leucoperm™ (BUF09)

For surface staining, block Fc receptors using Seroblock (mouse BUF041, or human BUF070)

Robust easy-to-use protocols (bio-rad-antibodies.com/fc-protocols) will give you the best staining. Identify your cells by following simple gating strategies (bio-rad-antibodies.com/gatingstrategies) to help you make the most of your data.

Before Ficoll spin

Blood

Ficoll

After Ficoll spin

Plasmaandplatelets

Ficoll

RBCs

PBMCs (interphase)

Granulocytes

Flow Cytometry - Steps to Success

Antigen specifi c protocols Analyze the data

Check your samples