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Protocol Compatibility Table for Immune Signaling and Phenotyping by Flow Cytometry

This protocol compatibility table identifies the surface and intracellular antibodies that work with a common protocol. Find additional details about the content and navigating the table in the text at the bottom of the page.

Antibody Clone Information and Protocol Compatibility Testing

Antibody Clone Information Protocol Compatibility Testing Available Conjugates
(Emission spectra (nm) listed below fluorophore name)
  Violet Laser Blue Laser Red Laser
  Protocols violet Fluor™ 450 Pacific Blue™ FITC Alexa Fluor® 488 PE PE-Cy®5 PerCP PerCP-Cy®5.5 PE-Cy®7 APC Alexa Fluor® 647 red Fluor™ 710 Alexa Fluor® 700 APC-Cy®7
Antibody
Epitope
Location
Species
Reactivity
1 2 3 4 450 455 520 520 578 667 678 695 785 660 668 710 719 785
Aiolos (D1C1E) IC H, M Not Recommended Recommended Recommended Not Tested       94568 21799           10896   59612  
Phospho-Akt
(Thr308) (D25E6)
IC H, M, R, Mk Not Recommended Recommended Recommended Not Tested       43506 13842           48646      
BCL6 (D4I2V) IC H Not Recommended Recommended Recommended Not Tested       26556 84882           41194      
Cleaved Caspase-3
(D3E9)
IC H, M Not Recommended Recommended Recommended Not Tested   8788   9603 12768           9602      
CD3 (17A2) EC M Recommended Decreased Performance Recommended Recommended 38527   86603   28306         24265   26582   17857
CD3 (UCHT1) EC H Recommended Recommended Recommended Recommended 61347   86774   46233     31767 62670 19881   44330   72359
CD4 (RM4-5) EC M Recommended Recommended Recommended Recommended 92599   96127   26589     49482 38341 82116   75508   46328
CD4 (RPA-T4) EC H Recommended Recommended Recommended Recommended 26755   48705   55927     46699 66182 78292       94467
CD8α (2.43) EC M Recommended Decreased Performance Recommended Recommended 17083   35467   56984     72461 87922 64786        
CD8α (RPA-T8) EC H Recommended Not Recommended Recommended Recommended 25109   55397   88829     75003 27442 64915   86135   26030
CD11b/ITGAM (M1/70) EC H, M Recommended Recommended Recommended Recommended 70078   24442   24965 16538   85601   41249   55274   81222
CD11c (3.9) EC H Decreased Performance Not Recommended Not Recommended Recommended 97473   69627   56025       80342 36268   42847   51562
CD14 (61D3) EC H Recommended Recommended Recommended Recommended 82944   29943   59896     99811 44947 36377   64342    
CD19 (1D3) EC M Recommended Not Recommended Not Recommended Decreased Performance 54508   53343   82168     43145 27221 39831   72880    
CD19 (SJ25C1) EC H Recommended Not Recommended Decreased Performance Recommended     49255   60751   47432   78813 31943        
CD24 (M1/69) EC M Recommended Not Recommended Not Recommended Decreased Performance 9705       90378         68390        
CD25/IL2-Rα (BC96) EC H Decreased Performance Not Recommended Not Recommended Not Recommended                 43212 20164        
CD27 (O323) EC H Recommended Not Recommended Not Recommended Decreased Performance 70511   35379   55584       66120 42404       19805
CD28 (CD28.2) EC H Recommended Not Recommended Not Recommended Not Recommended     15666   27826     25544 82076 73906        
CD38 (HIT2) EC H Recommended Not Recommended Not Recommended Not Recommended 24713   90005   90775     10284   73977        
CD44 (IM7) EC H, M Recommended Recommended Recommended Recommended 53289   75122   88151 94170   38200 43675 80813   63882   86109
CD45 (30-F11) EC M Recommended Recommended Recommended Recommended 83090   62307   47742       60943 41104   99856    
CD45 (HI30) EC H Recommended Recommended Recommended Recommended 74292   86532   89492     32125 28418 98228   39170   77322
CD45R/B220
(RA3-6B2)
EC H, M Recommended Recommended Recommended Recommended 67084   34399   93992 16935   17299 82922 98126   82984   31953
CD56/NCAM1 (MY31) EC H Recommended Not Recommended Not Recommended Decreased Performance     67184   73521       42804 51997        
CD62L/L-Selectin
(MEL-14)
EC M Recommended Not Recommended Not Recommended Decreased Performance     76378   32908     90088 61885 46592   60310   18118
Phospho-CD79A
(Tyr182) (D1B9)
IC H Not Recommended Recommended Recommended Not Tested       52821 14948           29742      
CD68 (D4B9C) EC H Decreased Performance Recommended Recommended Not Tested       24850 79594                  
CD86/B7-2 (GL-1) EC M Recommended Decreased Performance Decreased Performance Recommended     99879   60712       11527 84393        
CD161/NK1.1 (PK136) EC M Recommended Not Recommended Recommended Recommended     28486   45378     88957 78652 35986   19809    
CD161/KLRB1
(HP-3G10)
EC H Decreased Performance Not Recommended Not Recommended Not Recommended 26540   67086   83130     26217   51113        
Phospho-CREB
(Ser133) (87G3)
IC H, M, R Not Recommended Recommended Recommended Not Tested       9187 14228           14001      
EOMES (D8D1R) IC H Not Recommended Recommended Recommended Not Tested         17984           42582      
FoxP3 (3G3) IC M Not Recommended Not Recommended Not Recommended Recommended         65210       10680 41442        
F4/80 (BM8.1) EC M Recommended Decreased Performance Recommended Not Tested 40781   52267   64763     88154 80380 86007   94009   19233
GATA-3 (D13C9) IC H, M Not Recommended Recommended Recommended Not Tested         13411                  
Granzyme B (D2H2F) IC H, M Not Recommended Recommended Recommended Not Tested       33359 65563           50590      
Helios (D8W4X) IC H, M Not Recommended Recommended Not Tested Not Tested       56424 29360           65432      
Phospho-Histone
H2A.X (Ser139)
IC H, M, R, Mk Not Recommended Recommended Recommended Not Tested       9719 5763           9720      
Histone H3 (D1H2) IC H, M, R, Mk Not Recommended Recommended Decreased Performance Not Tested   12167     82241           12230      
Phospho-Histone H3
(Ser10) (D2C8)
IC H, M, R, Mk, Z Not Recommended Recommended Recommended Not Tested   8552   3465 5764           3458      
Acetyl-Histone H3
(Lys9) (C5B11)
IC H, M, R, Mk, Z Not Recommended Recommended Recommended Not Tested   11857   9683 28036           4484      
Tri-Methyl-Histone H3
(Lys27) (C36B11)
IC H, M, R, Mk Not Recommended Recommended Recommended Not Tested       5499 40724           12158      
Tri-Methyl-Histone H3
(Lys36) (D5A7)
IC H, M, R, Mk Not Recommended Recommended Not Recommended Not Tested         46287                  
Tri-Methyl-Histone H3
(Lys4) (C42D8)
IC H, M, R, Mk, Dm, Sc Not Recommended Recommended Recommended Not Tested                     12064      
HLA-DR (L243) EC H Recommended Recommended Recommended Not Recommended     54126   78397   17634         46861   92482
Ikaros (D6N9Y) IC H, M Not Recommended Recommended Recommended Not Tested       89389 33462           15740      
IκBα (L35A5) IC H, M, R, Mk, B, Pg Not Recommended Recommended Recommended Not Tested   13656   5743 7523                  
Phospho-IRF-3
(Ser396) (D6O1M)
IC H, M, R Not Recommended Recommended Not Recommended Not Tested       53539 83611                  
MHC Class II (I-A/I-E)
(M5/114.15.2)
EC M Recommended Recommended Recommended Recommended 86628   42594   67762     16114   64776   29097   55413
Phospho-NF-kB p65
(Ser536) (93H1)
IC H, M, R, Hm, Mk, Pg Not Recommended Recommended Recommended Not Tested       4886 5733           4887      
Nur77 (D63C5) IC H Not Recommended Recommended Recommended Not Tested         59999                  
Phospho-p44/42 MAPK (Erk1/2)
(Thr202/Tyr204) (197G2)
IC H, M, R, Mk, Mi, Dm, Z, Pg Not Recommended Recommended Recommended Not Tested   14196   13214 14095       98168 33370 13148   15892  
Phospho-PLCγ1
(Tyr783) (D6M9S)
IC H, M Not Recommended Recommended Not Tested Not Tested       25678 14461           88717      
Phospho-S6 Ribosomal Protein
(Ser235/236) (D57.2.2E)
IC H, M, R, Mk, Mi, Sc Not Recommended Recommended Recommended Not Tested   8520   4803 5316       34411 14733 4851   27036  
Phospho-SLP-76
(Ser376) (D7S1K)
IC H Not Recommended Recommended Not Recommended Not Tested   83768   90247 16318           40887      
Phospho-Stat1
(Tyr701) (58D6)
IC H, M Not Recommended Recommended Not Recommended Not Tested       9174 8062           8009      
Phospho-Stat2
(Tyr690) (D3P2P)
IC H, R Not Recommended Recommended Not Recommended Not Tested       68826 77366           90740      
Phospho-Stat3
(Ser727)(D4X3C)
IC H Not Recommended Recommended Not Recommended Not Tested       58978 87544           71958      
Phospho-Stat3
(Tyr705) (D3A7)
IC H, M, R, Mk Not Recommended Recommended Not Recommended Not Tested       4323 8119           4324      
Phospho-Stat4
(Tyr693) (D2E4)
IC H Not Recommended Recommended Not Recommended Not Tested         13223                  
Phospho-Stat5
(Tyr694) (C71E5)
IC H, M Not Recommended Recommended Not Recommended Not Tested       3939 5387           9365      
Phospho-Stat6
(Tyr641)(D8S9Y)
IC H, M Not Recommended Recommended Not Recommended Not Tested         99902           10205      
Phospho-STING
(Ser365) (D1C4T)
IC M Not Recommended Recommended Recommended Not Tested                            
Phospho-STING
(Ser366) (D8K6H)
IC H Not Recommended Recommended Recommended Not Tested       41622             43499      
Phospho-Syk
(Tyr525/526) (C87C1)
IC H Not Recommended Recommended Recommended Not Tested       4349 6485           12081      
Phospho-TBK1/NAK
(Ser172) (D52C2)
IC H, M Not Recommended Recommended Recommended Not Tested       14586 13498           14590      
T-bet/TBX21 (D6N8B) IC H Not Recommended Recommended Recommended Not Tested       14298             14307      
TCF1/TCF7 (C63D9) IC H, M Not Recommended Recommended Recommended Not Tested   9066   6444 14456           6709      
Phospho-Zap-70 (Tyr319)/Syk
(Tyr352) (65E4)
IC H, M Not Recommended Recommended Not Tested Not Tested       73382 14791           82975      
Phospho-4E-BP1
(Thr37/46) (236B4)
IC H, M, R, Mk, Dm Not Recommended Recommended Recommended Not Tested       2846 7547           5123      

Table Key:   IC: Intracellular   EC: Extracellular   Recommended Recommended   Decreased Performance Decreased Performance   Not Recommended Not Recommended   Not Tested Not Tested

Species Reactivity Key:  H—Human  M—Mouse  R—Rat  Mk—Monkey  B—Bovine  Pg—Pig  Mi—Mink  Dm—D. melanogaster  Z—Zebrafish  Sc—S. cerevisiae

Protocol Overview

Protocol No. Protocol Information Brief Description of Staining Preparation Protocol Available Fixation-Permeabilization Kits
1 Live Cell Surface Staining Protocol Protocol where there is no fixation or permeabilization of the cells before staining Not Applicable
2 Fixed Cell Intracellular Staining – Methanol Permeabilization Protocol Uses formaldehyde and methanol for fixation and permeabilization of the cells before staining 13593
3 Fixed Cell Intracellular Staining – Triton™ X-100 Permeabilization Protocol Uses formaldehyde and Triton™ X-100 for fixation and permeabilization of the cells prior to staining 51995
4 Fixed Cell Intracellular Staining – FoxP3/Transcription Factor Protocol Fixation and permeabilization buffer set optimized for staining with antibodies to transcription factors and nuclear proteins 43481

Flow Cytometry - An Antibody and Protocol Selection Guide for Establishing a Multicolor Panel

There are many different protocols for you to choose from when staining either live or fixed cells in a flow cytometry experiment. Your protocols must be compatible with the antibodies used to stain the cells to avoid negatively affecting the epitope recognition while still allowing the antibody to access the cellular component where the protein of interest resides.

For example, staining intracellular targets for phospho-flow cytometry experiments requires that you fix and permeabilize your cells before staining. However, fixation (fix) and permeabilization (perm) protocols can alter the interaction between epitope and antibody. In particular, the choice of permeabilization reagent can have a significant influence on the sensitivity, specificity, and functionality of antibodies used within the assay. This can be both antigen- and clone-dependent.

Permeabilization is commonly achieved using solvents such as methanol or detergents like Triton™ X-100 or saponin. Saponin offers the least thorough permeabilization; it forms pores in the plasma membrane by associating with cholesterol but does not permeabilize intracellular membranes, such as the nuclear membrane or mitochondrial membrane. Triton™ X-100 and methanol will adequately permeabilize most intracellular membranes, with methanol providing the most thorough permeabilization. However, methanol can be disruptive to epitopes, and some antibodies, including some surface markers, will not work following methanol permeabilization.

The Protocol Compatibility Table Identifies the Surface and Intracellular Antibodies that Work with a Common Protocol

Cell Signaling Technology® (CST) offers a diverse catalog of flow-cytometry validated antibodies conjugated with different fluorescent dyes. We’ve put together this Protocol Compatibility Table for Immune Signaling and Phenotyping by Flow Cytometry to help you quickly identify which staining protocols antibodies in the CST catalog are compatible with, especially when selecting multiple antibodies to use together in multiplex panels for signaling flow cytometry. Specifically, this table will help you understand:

  • which surface marker antibodies (CD markers) are compatible only with live-cell protocols
  • which CD markers work in fixation-permeabilization protocols typically used for intracellular staining flow cytometry
  • which fixation and permeabilization protocols (fix-perm) to use with fix-perm compatible antibodies
  • which antibodies ONLY work with fix-perm protocols, and which of these protocols they work with

Navigating the Protocol Compatibility Table

Antibody Clone Information lists the CST antibody clone, epitope location, and species reactivity information for potential antibodies for your experiment.

Protocol Compatibility Testing identifies which of the most popular protocols were tested for each antibody clone to help you save time when building your panel. When selecting antibodies to multiplex in an experiment, you’ll want to make sure each clone is compatible with the same protocol. A green mark indicates the protocol is recommended, a yellow mark indicates the protocol was tested but may show decreased performance so further protocol optimization is recommended, a red mark indicates that the antibody is not recommended for that specific protocol, and a white mark means the protocol hasn’t been tested yet.

Available Conjugates lists the direct conjugate antibody products available from CST for each clone, organized from left to right by the approximate maximum emission of the respective fluorophore. Click on the linked product number to learn more about the specific antibody conjugate of interest.

For Research Use Only. Not For Use In Diagnostic Procedures.
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