Insight
Issue
4
Discover the different stages of cell differentiation Previewing Abcam tools for stem cell research
Tips to build multicolor panels for flow cytometry www.abcam.com/flow-tips
Stem cell markers Browse all stem cell products
Major embryonic stem cell markers Cell Surface Pluripotency Markers Target
Uniprot
Primary antibody
Cripto-1
P13385
PRF
Dax1
P51843
PRF
GDF1
P27539
PRF
GDF3
Q9NR23
PRF
Nanog
Q9H9S0
PRF
Oct4
Q01860
PRF
Sall4
Q9UJQ4
PRF
Induced (pluripotent stem) PS cell markers
Immunoassays
Proteins
Target
Uniprot
Primary antibody
Immunoassays
Proteins
ELISA SSE
RA
CD13
P15144
PRF
ELISA SSE
RA
R
Nanog
Q9H9S0
PRF
ELISA SSE
RA
R RA
Germ cell markers during specification
ELISA SSE
RA
c-kit
P10721
PRF
ELISA SSE
RA
Dazl
Q92904
PRF
R
MVH
Q9NQI0
PRF
RA
Oct4
Q01860
PRF
PRDM1/Blimp1
O75626
PRF
Sox2
P48431
PRF
ELISA SSE
Tbx3
O15119
PRF
R
Tcl1
P56279
PRF
R
ELISA SSE
RA R R
ELISA SSE
RA R
The ectoderm, and endoderm markers Transcription Factors
FGF-8
P55075
P
RA
RA
Noggin
Q13253
PRF
RA
RA
Otx2
P32243
PRF
R
PRF
R
p63/TP73L
Q9H3D4
PRF
PRF
RA
Pax2
Q02962
PRF
R
RA
Pax6
P26367
PRF
R
c-Myc
P01106
PRF
Klf4
O43474
PRF
Mycn
P04198
Nanog
Q9H9S0
Oct4
Q01860
PRF
Sall4
Q9UJQ4
PRF
Sox18
P35713
PRF
Sox2
P48431
PRF
Tcl1
P56279
PRF
ELISA SSE
ELISA SSE
ELISA SSE
R R ELISA SSE
RA R
Major hematopoietic stem cell markers
Major cancer stem cell markers
Target
Uniprot
Primary antibody
Immunoassays
Proteins
Target
Uniprot
Primary antibody
Immunoassays
Proteins
CD4
P01730
PRF
ELISA SSE
RA
ALDH1
P00352
PRF
ELISA SSE
R
CD34
P28906
PRF
R
CD13
P15144
PRF
ELISA SSE
RA
CD38
P28907
PRF
R
CD24
P25063
PRF
CD48
P09326
PRF
RA
CD44
P16070
PRF
CD90 / Thy1
P04216
PRF
R
CD90 / Thy1
P04216
P
R
CD244
Q9BZW8
P
ELISA
RA
CD133
O43490
PRF
R
c-kit
P10721
PRF
ELISA SSE
RA
CK5
P13647
PRF
Flt-3/CD135
P36888
PRF
ELISA SSE
RA
CK8
P05787
PRF
Lin28
Q9H9Z2
PRF
RA
CK10
P13645
PRF
Mac-1/CD11b
P11215
PRF
R
CK14
P02533
PRF
Slamf1/CD150
Q13291
PRF
R
CK15
P19012
PRF
CK18
P05783
PRF
Integrin alpha 6/CD49f
P23229
PRF
p75 NGFR/CD271
P08138
PRF
ELISA
R ELISA
ELISA SSE
R
R R R
ELISA SSE
R R
ELISA SSE
R R
ELISA
A: Active; ELISA: ELISA kit; F: BSA and azide free (± low endotoxin tested); P: Primary antibody; R: Recombinant; R: Recombinant monoclonal; SSE: SimpleStep ELISA® Kit
RA
Major cardiac stem cell markers Target
Uniprot
Primary antibody
67LR/RPSA
P08865
PRF
Cardiac troponin I
P19429
PRF
ELISA SSE
CD31
P16284
PRF
ELISA SSE
CD34
P28906
CD45
P08575
CD90 / Thy1
Major osteoprogenitor cell markers Proteins
Target
Uniprot
Primary antibody
Immunoassays
Proteins
R
ALPP
P05187
PRF
ELISA SSE
R
R
BAP1
Q92560
PRF
RA
RA
BAP31
P51572
PRF
R
PRF
R
BMP-2
P12643
PRF
PRF
RA
CD146/MUC18
P43121
PRF
P04216
PRF
R
Collagen I
P02452
PRF
CD105
P17813
PRF
RA
Collagen II
P02458
PRF
CD133
O43490
PRF
R
Decorin
P07585
P
ELISA SSE
RA
c-kit
P10721
PRF
RA
DKK3
Q9UBP4
PRF
ELISA SSE
RA
Integrin beta-1/CD29
P05556
PRF
R
FGF-2
P09038
PRF
ELISA
RA
Isl1
P61371
PRF
R
Fibronectin
P02751
PRF
ELISA SSE
R
Nkx2.5
P52952
PRF
R
Gremlin 1
O60565
P
Oct4
Q01860
PRF
ELISA SSE
RA
IGFBP-3
P17936
PRF
ELISA SSE
RA
TGF-β1
P01137
PRF
ELISA
RA
M-CSF
P09603
PRF
ELISA SSE
RA
VEGFR2/KDR
P35968
PRF
ELISA SSE
RA
Osteocalcin
P02818
PRF
ELISA SSE
R
Osteopontin
P10451
PRF
ELISA SSE
R
RANKL
O14788
PF
ELISA
RA
RUNX2
Q13950
PRF
Proteins
Sclerostin
Q9BQB4
P
RA
Sp7/Osterix
Q8TDD2
PRF
RA
SPARC
P09486
PRF
ELISA SSE
RA
RA
TGF-β1
P01137
PRF
ELISA
RA
R
Tpo
P07202
PRF
ELISA SSE
RA
Immunoassays
ELISA SSE
ELISA SSE
Major mesenchymal stem cell markers Target
Uniprot
Primary antibody
CD10/Neprilysin
P08473
PRF
CD13
P15144
PRF
CD73
P21589
PRF
Immunoassays ELISA SSE
ELISA
CD90 / Thy1
P04216
PRF
CD105
P17813
PRF
ELISA SSE
RA
CD140b/PDGFRB
P09619
PRF
ELISA
RA
CD146/MUC18
P43121
PRF
HER-2/erbB2 (CD340)
P04626
PRF
Integrin beta-1/CD29
P05556
PRF
Oct4
Q01860
PRF
p75 NGFR/CD271
P08138
PRF
R
Target
Uniprot
alpha-smooth muscle actin
P12814
PRF
CD56/NCAM1
P13591
PRF
CD146/MUC18
P43121
PRF
Desmin
P17661
PRF
FABP3
P05413
PRF
Myosin-9
P35579
PRF
VE-Cadherin
P33151
PRF
RA R
ELISA SSE
R R
RA
R ELISA SSE
RA R
Major skin stem cell markers
RA
Target
Uniprot
Primary antibody
R
CD34
P28906
PRF
ELISA SSE
RA
CK15
P19012
PRF
ELISA
RA
DLL1
O00548
P
ELISA SSE
R
EGFR
P00533
PRF
ELISA SSE
RA
Follistatin
P19883
PRF
ELISA SSE
RA
IGF1R/CD221
P08069
PRF
ELISA
RA
P23229
PRF
ELISA
Major myogenic precursor cell markers Primary antibody
ELISA
Immunoassays
Proteins R R
Immunoassays
Proteins
Integrin alpha 6/CD49f
ELISA SSE
R
p63/TP73L
Q9H3D4
PRF
ELISA SSE
Tenascin C
P24821
PRF
ELISA
R
TGFBR2/TBRII
P37173
PRF
ELISA
R
ELISA
R
R
R R ELISA SSE
R R
ELISA SSE
R
Major neural stem cell markers Target
Uniprot
Primary antibody
ABCG2/CD338
Q9UNQ0
PRF
R
CD24
P25063
PRF
R
CD34
P28906
PRF
R
CD45
P08575
PRF
RA
CD133
O43490
PRF
R
FGFR4
P22455
PRF
RA
Sox2
P48431
PRF
RA
Immunoassays
Proteins
Major adipose-derived stem cell markers Target
Uniprot
Primary antibody
CD34
P28906
PRF
CD44
P16070
PRF
ELISA
R
ICAM-1/CD54
P05362
PRF
ELISA SSE
RA
Immunoassays
Proteins R
Major intestinal stem cell markers Target
Uniprot
Primary antibody
Bmi1
P35226
PRF
R
Gremlin 1
O60565
P
RA
Hopx
Q9BPY8
PRF
R
Tert
O14746
PRF
R
Immunoassays
A: Active; ELISA: ELISA kit; F: BSA and azide free (± low endotoxin tested); P: Primary antibody; R: Recombinant; R: Recombinant monoclonal; SSE: SimpleStep ELISA® Kit
Proteins
Select the right antibody for working with collagen Explore our protocol tips for working with collagen
Collagens are highly conserved proteins, characterized by an uninterrupted glycine-X-Y triplet repeat that gives them their triple helical structure. It is the close similarity of this triple helical structure across the different collagen types that can make it very difficult to generate antibodies specific for a particular collagen. In order to make a collagen antibody specific, the immunogen used for the antibody production must either be dependent on non-denatured three-dimensional collagen epitopes or shorter peptide sequences only found in specific collagens. Depending on the type of antibody you use, you will need to adapt your protocol accordingly.
Structure
Distribution
Sequence specific antibodies
I
Fibrillar
Ubiquitous
ab138492
ab34710
Collagen
Structure-specific antibodies
II
Fibrillar
Cartilage, vitreous
ab188570
ab34712
III
Fibrillar
Skin, blood vessels, intestine
ab184993
ab7778
IV
Network
Basement membranes
ab214417
ab6586
V
Fibrillar
Ubiquitous
VI
Network
Ubiquitous
ab182744
ab7046 ab6588
VII
Anchoring Fibrils
Dermis, bladder
ab223639
ab93350
ab58776
VIII
Network
Ubiquitous
IX
FACIT
Cartilage, cornea, vitreous
X
Network
Cartilage
ab182563
ab134568
XI
Fibrillar
Cartilage, intervertebal disc
ab196613
XV
FACIT
Basement membranes
ab58717
XVII
MACIT
Hemidesmo-somes in epithelia
ab184996
ab58632
ab79878
Table 1. Vertebrate collagens. 1Adapted from Shoulders MD, Raines RT. COLLAGEN STRUCTURE AND STABILITY. Annual review of biochemistry. 2009;78:929958. doi:10.1146/annurev.biochem.77.032207.120833.
References 1. Shoulders MD, Raines RT. COLLAGEN STRUCTURE AND STABILITY. Annual review of biochemistry. 2009;78:929-958. doi:10.1146/annurev.biochem.77.032207.120833. 2. Mocan E.Tagadiuc O.Nacu V. (2011) Aspects of collagen isolation procedure. Clin. Res. Studies 2, 3–5
Stem Cell marker antibody panel View our stem cell support page Product code
Panel name
Component product code ab16287
ab109884
ab238602
ab107156
ab254022
ab93758
ab93759
ab254027
ab254028
Embryonic Stem Cell Marker – Human I
Embryonic Stem Cell Marker – Human II
Embryonic Stem Cell Marker – Mouse
Hematopoietic Stem Cell Marker Panel - Human
Mesenchymal Stromal Cell Marker - Human
Mesenchymal Stromal Cell Marker - Mouse
Neural Stem Cell Marker - Human
Neural Stem Cell Marker Panel Mouse
Size
Component name
Tested applications
Species reactivity
1 x 50 µl
Anti-SSEA4 antibody [MC813]
Flow Cyt,ICC/IF,IHC-FoFr,WB
Hu,Ms,Rat Hu,Rb
ab16288
1 x 50 µg
Anti-TRA-1-60 (R) antibody [TRA-1-60]
Flow Cyt,ICC,ICC/IF,IHC-Fr,IP, RIA,WB
ab19857
1 x 50 µg
Anti-Oct4 antibody - ChIP Grade
ChIP,CHIPseq,Flow Cyt,ICC/IF,IP,WB
Hu,Ms,RMk,Shp Hu
ab21624
1 x 50 µl
Anti-Nanog antibody - ChIP Grade
ChIP,Flow Cyt,ICC,ICC/IF,IHC-P, sELISA,WB
ab97959
1 x 50 µg
Anti-SOX2 antibody
IHC - Wmt,ICC/IF,IHC-P,IHC-Fr, IP,WB
Chk,Hrs,Hu,Ms,Pig,Ql, Rat,Rtrout,Sc,Tray,Xl,Zfsh
ab21624
1 x 50 µl
Anti-Nanog antibody - ChIP Grade
ChIP,Flow Cyt,ICC,ICC/IF,IHC-P, sELISA,WB
Hu
ab19857
1 x 50 µg
Anti-Oct4 antibody - ChIP Grade
ChIP,CHIPseq,Flow Cyt,ICC/IF,IP,WB
Hu,Ms,RMk,Shp
Chk,Hrs,Hu,Ms,Pig,Ql, Rat,Rtrout,Sc,Tray,Xl,Zfsh
ab97959
1 x 50 µg
Anti-SOX2 antibody
IHC-Wmt,ICC/IF,IHC-P,IHC-Fr, IP,WB
ab16288
1 x 50 µg
Anti-TRA-1-60 (R) antibody [TRA-1-60]
Flow Cyt,ICC,ICC/IF,IHC-Fr,IP, RIA,WB
Hu,Rb
ab16285
1 x 50 µl
Anti-SSEA1 antibody [MC-480]
Flow Cyt,ICC/IF,IHC-FrFl,IHC-P, IHC-FoFr,IP
Hu,Ms
ab19857
1 x 50 µg
Anti-Oct4 antibody - ChIP Grade
ChIP,CHIPseq,Flow Cyt,ICC/IF, IP,WB
Hu,Ms,RMk,Shp
ab80892
1 x 50 µl
Anti-Nanog antibody
Flow Cyt,IHC - Wmt,ICC,ICC/IF, IHC-P,WB
Ms
ab97959
1 x 50 µg
Anti-SOX2 antibody
IHC-Wmt,ICC/IF,IHC-P,IHC-Fr,IP,WB
Chk,Hrs,Hu,Ms,Pig,Ql, Rat,Rtrout,Sc,Tray,Xl,Zfsh
ab81289
2 x 10 µl
Anti-CD34 antibody [EP373Y]
Flow Cyt,ICC/IF,IHC-Fr,IHC-P,IP,WB
EliAB,Dog,Hu,Ms,Rat,Shp
ab108403
2 x 10 µl
Anti-CD38 antibody [EPR4106]
WB, IHC-P, Flow Cyt
Hu
ab133707
2 x 10 µl
Anti-CD59 antibody [EPR6425(2)]
WB, IHC-P
Hu
ab133350
2 x 10 µl
Anti-CD90 / Thy1 antibody [EPR3133]
IHC-P, ICC/IF, WB
Hu
ab32363
2 x 10 µl
Anti-c-Kit antibody [YR145]
IHC-P, WB
Cm,Hu
ab6124
1 x 50 µg
Anti-CD44 antibody [F10-44-2]
Flow Cyt,ICC/IF,IHC-P,IHC-Fr,IP,WB
Hu
ab10559
1 x 50 µg
Anti-CD45 antibody
Flow Cyt,ICC/IF,IHC-P,IHC-Fr,WB
Hu,Mk,Pig
ab23894
1 x 50 µg
Anti-CD90 / Thy1 antibody [AF-9]
ELISA,Flow Cyt,IHC-Fr
Hu
ab44967
1 x 50 µl
Anti-CD105 antibody [105C02]
Flow Cyt,IHC-P,IHC-Fr,WB
Hu
ab52971
1 x 10 µl
IHC-P,IHC-Fr,WB
Hu
ab3105
1 x 50 µg
Anti-CD90 / Thy1 antibody [IBL-6/23]
Flow Cyt,ICC/IF,IHC-P,IHC-Fr, IHC-FoFr,IP
Ms
ab25340
1 x 50 µg
Anti-CD44 antibody [KM201]
BL,Flow Cyt,IHC-P,IHC-Fr, IHC-FoFr,IP,WB
Ms
ab25386
1 x 50 µg
Anti-CD45 antibody [I3/2.3]
Flow Cyt,IHC-P,IHC-Fr,IP
Ms
ab51317
1 x 50 µg
Anti-Sca1 / Ly6A/E antibody [E13 161-7]
ICC/IF
Ms
ab95623
1 x 50 µg
Anti-Integrin beta 1 antibody [KMI6]
Flow Cyt,ICC/IF,IHC-Fr,IP,WB
Ms
ab105389
1 x 20 µL
Anti-Nestin antibody [SP103]
Flow Cyt,ICC/IF,IHC-P,WB
Hu
ICC/IF,IHC-P,IHC - Wmt, Sandwich ELISA,WB
Hu,Leucor eri,Ms
Flow Cyt,ICC/IF,IHC-P,IP,WB
Dog,Hu,Ms,Rat
KO VALIDATED
KO VALIDATED
KO VALIDATED
Anti-Integrin beta 1 antibody [EP1041Y] KO VALIDATED
KO VALIDATED
ab92494
1 x 20 µL
Anti-SOX2 antibody [EPR3131]
ab216327
1 x 20 µL
Anti-Occludin antibody [EPR20992]
ab40772
1 x 20 µL
Anti-E Cadherin antibody [EP700Y]
Flow Cyt,ICC/IF,IHC-Fr,IHC-P,WB
Hu
ab108937
1 x 20 µL
Anti-Hes1 antibody [EPR4226]
Flow Cyt,ICC/IF,IHC-P,WB
Hu,Ms,Rat
ab52627
1 x 20 µL
Anti-Notch1 antibody [EP1238Y]
Flow Cyt,ICC/IF,IHC-P,WB
Hu,Ms,Cow
ab11512
2 x 10 µg
Anti-E Cadherin antibody [DECMA-1]
Flow Cyt,ICC/IF,IHC-Fr,WB
Dog,Ms
ab108937
2 x 10 µl
Anti-Hes1 antibody [EPR4226]
Flow Cyt,ICC/IF,IHC-P,WB
Hu,Ms,Rat
ab6142
2 x 10 µg
Anti-Nestin antibody [Rat 401]
ICC/IF,IHC-Fr,IHC-FoFr,IHC-P,WB
Cat,Hu,Mk,Shp
ab52627
2 x 10 µl
Anti-Notch1 antibody [EP1238Y]
Flow Cyt,ICC/IF,IHC-P,WB
Cow,Hu,Ms
ab216327
2 x 10 µl
Anti-Occludin antibody [EPR20992]
Flow Cyt,ICC/IF,IHC-P,IP,WB
Dog,Hu,Ms,Rat
ICC/IF,IHC-P,IHC - Wmt, Sandwich ELISA,WB
Hu,Leucor eri,Ms
ab92494
2 x 10 µl
Anti-SOX2 antibody [EPR3131]
KO VALIDATED
KO VALIDATED
Chk: Chicken; Cm: Common marmoset; EliAB: African bush elephant; Hrs: Horse; Hu: Human; Leucor eri: Leucoraja erinacea; Ms: Mouse; Ql: Quail; Rb: Rabbit; RMk: Rhesus monkey; Rtrout: Rainbow trout; Sc: Spotted catshark; Shp: Sheep; Tray:Thornback ray; Xl: Xenopus laevis; Zfsh: Zebrafish
Antibody selection and enhanced validation strategies webinar Speaker: Alejandra Solache, Vice President, New Product Development, Abcam plc
Antibody reproducibility remains an important issue across the Life Sciences industry. This webinar will cover: Advantages and constraints of available antibody formats Guidance on antibody selection Industry best practice for antibody validation The importance of validating antibodies for specific applications
View the webinar
Fluorescent staining of viable cells Visit our complete dye review online
Amine-reactive dyes for live:dead cell assays Amine-reactive dyes weakly stain viable cells by binding to cell surface amines and strongly stain membrane-compromised cells by reacting with intracellular amines. Dead and live cells can be differentiated by fluorescence level. Assay
Amine-reactive dyes
Instrument
Flow cytometer
Notes
Assay kits
Ex/Em 410/450 nm. Fixation compatible
ab176738
Ex/Em 408/512 nm. Fixation compatible
ab176739
Ex/Em 398/550 nm. Fixation compatible
ab176740
Ex/Em 353/442 nm. Fixation compatible
ab176741
Ex/Em 498/521 nm. Fixation compatible
ab176742
Ex/Em 547/573 nm. Fixation compatible
ab176743
Ex/Em 583/603 nm. Fixation compatible
ab176744
Ex/Em 649/660 nm. Fixation compatible
ab176745
Dye dilution assays The dyes in dye dilution assays are retained within cells over multiple generations. Daughter cells receive half of the dye of parent cells and assays are analyzed on a flow cytometer. Carboxyfluorescein succinimidyl ester (CFSE) is the longest established dye. Assay
Instrument
CFSE
Flow cytometer
CytoLabel Blue CytoLabel Green CytoLabel Red
Flow cytometer, microscope
CytoLabel Orange
Notes
Assay kits
Ex/Em 492/517 nm. Cytotoxic at higher concentrations. ab113853 Ex/Em 403/454 nm
ab176726
Ex/Em 511/525 nm
ab176735
Ex/Em 628/643 nm
ab176736
Ex/Em 542/556 nm
ab176737
Live Cell Labeling Kit The kits are a set of tools used to label cells for fluorescence microscopic investigations and flow cytometric investigations of cellular functions. The effective labelling of cells provides a powerful method for studying cellular events in a spatial and temporal context. Detection
Instrument
Red Fluorescence Blue Fluorescence Blue Fluorescence (Ex405nm) Green Fluorescence Green Fluorescence (Ex405nm)
Flow cytometer, fluorescence microscope
Notes
Assay kits
Assay time 2h. Ex/Em = 649/659 nm
ab187965
Assay time 2h. Ex/Em = 360/445 nm
ab187963
Assay time 2h. Ex/Em = 410/450 nm
ab187966
Assay time 2h. Ex/Em = 495/515 nm
ab187964
Assay time 2h. Ex/Em = 410/500 nm
ab187967
Directly conjugated antibodies for immunofluorescence and flow cytometry Explore our primary conjugates for flow or ICC/IF Recombinant RabMAb® technology provides excellent affinity and specificity In-house manufacturing ensures high-quality conjugates Individual product validation in 5-8 applications, including batch-validation Removal of free dye to ensure high signal to noise ratio Supporting data including images, references, and customer reviews
Fluorescent-labeled primary antibodies: Label
Validation data
Alexa Fluor
Immunofluorescence (ICC/IF), immunohistochemistry (IHC), flow cytometry
Phycoerythrin (R-PE)
ICC/IF and/or flow cytometry
FITC (Fluorescein isothiocyanate)
ICC/IF and/or flow cytometry
APC (Allophycocyanin)
ICC/IF and/or flow cytometry
PerCP (Peridinin chlorophyll)
ICC/IF and/or flow cytometry
®
Product highlight: Recombinant Anti-PD-L1 antibody [28-8] (Alexa Fluor® 488) (ab209959)
Anti-alpha Tubulin antibody [DM1A] Microtubule Marker (FITC) (ab64503)
Rabbit monoclonal antibody with high affinity and high specificity. Validated in Flow Cytometry and ICC/IF. Tested in Human.
Validated in IHC-Fr, Flow Cyt, ICC/IF. Tested in Mouse, Rat, Chicken, Guinea pig, Cow, Dog, Human, Pig, Xenopus laevis, Gerbil. Cited in 12 publication(s).
Ab209959 staining PDL1 in CHO-PDL1 cells. The lower panels demonstrate that ab209959 does not cross react with un-transfected CHO cells.
ICC/IF image of ab64503 stained human HeLa cells. Alexa Fluor® 594 Wheat Germ Agglutinin conjugate was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).
Alexa Fluor® is a registered trademark of Life Technologies. Alexa Fluor® dye conjugates contain(s) technology licensed to Abcam by Life Technologies.
Multicolor flow cytometry panel design Download fluorochrome chart Our guide to help you build successful multi-color flow cytometry panels 1. Know your flow cytometer Lasers: only fluorochromes excited by the corresponding wavelength of light from the laser can be used
Filters: detection of light emitted from fluorochromes is controlled by filters
UV 355 nm Violet 405 nm
<575 nm detected
Blue 488 nm
To ensure optimal detection, make sure you understand the combination of lasers /filters on your machine. Refer to your instrumentâ&#x20AC;&#x2122;s manual or speak to your core facility
Yellow 561 nm
>520 nm detected 520 LP
630/20 (= 630Âą10nm)
Long pass dichroic filter
Band pass filter
575 SP >575 nm reflected
Red 640 nm Far red 750 nm
620-640 nm detected
<520 nm reflected
Short pass dichroic filter
2. Know your cell population, antigens, and fluorochromes High antigen expression and/or high cell populations = use dimmer fluorochromes, eg PerCP
Low/unknown antigen expression and/or low cell populations = use brighter fluorochromes, eg PE
Low
High
Bright
Dim
Check the relative brightness of your fluorochromes at www.abcam.com/fluorochrome-chart
3. Minimize spectral overlap Overlap
Minimize emission spectra overlap
No overlap
Sacrifice bright fluorochromes to avoid overlap Compensation can be used to control the effects of spectral overlap
4. Include controls
Unstained cells for defining negative populations, cell size, and granularity
Live/dead markers to isolate healthy cells
Single-stained positive controls for setting compensation
Fluorescence minus one staining to define positive populations
5. Optimize your staining protocol Antibody + Fc blocking reagent
Antibody Good titration
Bad titration
Antibody concentration: titrate your antibodies to avoid non-specific binding or reduced sensitivity www.abcam.com/multicolorflow
Specific antibody binding
Non-specific antibody binding
Fc blocking reagents: Human IgG for human Anti-CD16+ CD32 for mouse
Fc blocking: use Fc blocking reagents in cells with high content of Fc receptors (eg phagocytic cells) to avoid non-specific binding
How do you write a scientific paper in no time at all and not bore people to death? Never use a metaphor, simile, or another figure of speech which you are used to seeing in print. Never use a long word where a short one will do. If it is possible to cut a word out, always cut it out. Never use the passive where you can use the active. Never use a foreign phrase, a scientific word, or a jargon word if you can think of an everyday English equivalent. Break any of these rules sooner than say anything outright barbarous
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