CST Guide:

Section II: ANTIBODY APPLICATIONS
chapter 10: Flow Cytometry (F)
PE conjugated
antibodies deliver
robust Stoke’s
shifts and are ideal
for multiplexing.
Events
Phospho-Akt (Ser473)
Anti-rabbit IgG (H+L), F(ab’) 2
Fragment (PE Conjugate) #8885:
Flow cytometric analysis of Jurkat
cells, untreated (green) or treated
with LY294002 #9901, Wortmannin
#9951, and U0126 #9903 (blue), using
Phospho-Akt (Ser473) (D9E) XP ® Rabbit
mAb #4060 detected with #8885.
Flow Cytometry General Protocol
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized or equivalent grade water (dH 2 0).
1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH 2 O, mix.
2. 16% Formaldehyde (methanol free)
3. 100% Methanol
4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
5. Secondary Antibodies: Anti-mouse (#4408, #8890, #4410, #8887), Anti-rabbit (#4412, #8889, #4414, #8885), Anti-rat
(#4416, #4418)
B. Fixation
1. Collect cells by centrifugation and aspirate supernatant.
2. Resuspend cells in 0.5–1 ml 1X PBS. Add appropriate volume of formaldehyde to obtain a final concentration of
4% (e.g. 750 µl 1X PBS + 250 µl 16% formaldehyde).
3. Fix for 10 min in a 37°C water bath.
4. Chill tubes on ice for 1 min.
5. For extracellular staining with antibodies that do not require permeabilization, proceed to immunostaining (Section D)
or store cells in 1X PBS with 0.1% sodium azide at 4°C; for intracellular staining, proceed to permeabilization (Section C).
C. Permeabilization
NOTE: This step is critical for many CST antibodies.
1. Add ice-cold 100% methanol drop-wise to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol.
Alternatively, remove fix prior to permeabilization by centrifugation and resuspend in 90% methanol as described above.
2. Permeabilize for a minimum of 10 min on ice.
3. Proceed with Immunostaining (Section D) or store cells at -20°C in 90% methanol.
B. Preparation of Whole Blood (fixation, lysis, and permeabilization) for Immunostaining
1. Aliquot 100 μl fresh whole blood per assay tube.
2. OPTIONAL: Place tubes in rack in 37°C water bath for short-term treatments with ligands, inhibitors, drugs, etc.
3. Add 65 μl of 10% formaldehyde to each tube.
4. Vortex briefly and let stand for 15 min at room temperature.
5. Add 1 ml of 0.1% Triton X-100 to each tube.
6. Vortex and let stand for 30 min at room temperature.
7. Add 1 ml incubation buffer.
8. Pellet cells by centrifugation and aspirate supernatant.
9. Repeat Steps 7 and 8.
10. Resuspend cells in ice-cold 50% methanol in PBS (store methanol solution at -20°C until use).
11. Incubate at least 10 min on ice.
12. Proceed with staining or store cells at -20°C in 50% methanol.
C. Staining Using Unlabeled Primary and Conjugated Secondary Antibodies
NOTE: Account for isotype-matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies.
1. Add 2–3 ml incubation buffer to each tube and wash by centrifugation. Repeat.
2. Add primary antibodies diluted as recommended on datasheet or product webpage in incubation buffer.
3. Incubate for 30–60 min at room temperature.
4. Wash by centrifugation in 2–3 ml incubation buffer.
5. Resuspend cells in fluorochrome-conjugated secondary antibody diluted in incubation buffer according to the manufacturer’s
recommendations.
6. Incubate for 30 min at room temperature.
7. Wash by centrifugation in 2–3 ml incubation buffer.
8. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.
Cyclin B1
Protocol Reference: Chow, S.,
Hedley, D., Grom, P., Magari, R.,
Jacobberger, J.W., and Shankey,
T.V. (2005) Whole blood fixation
and permeabilization protocol
with red blood cell lysis for flow
cytometry of intracellular phosphorylated
epitopes in leukocyte
subpopulations. Cytometry A.
67, 4–17.
PI/RNase Staining
Solution labels DNA
content without RNA
crossreactivity.
10 4
10 3
10 2
10 1
R-Phycoerythrin
(PE) Conjugates
For the most up-to-date list of ready
to use flow validated antibodies conjugated
to PE, eliminating the time and
costs associated with PE conjugation
and subsequent validation, go to
www.cellsignal.com/peconjugates
D. Immunostaining
1. Aliquot 0.5–1x10 6 cells into each assay tube (by volume).
2. Add 2 ml incubation buffer to each tube and wash by centrifugation. Repeat.
3. Resuspend cells in 100 µl of diluted primary antibody prepared in incubation buffer at the recommended dilution.
See individual antibody datasheet or product webpage for the appropriate dilution.
4. Incubate for 1 hr at room temperature.
5. Wash by centrifugation in 2 ml incubation buffer.
6. If using a fluorochrome-conjugated primary antibody, resuspend cells in 0.5 ml 1X PBS and analyze on flow cytometer;
for unconjugated or biotinylated primary antibodies, proceed to immunostaining (Step 9).
7. Resuspend cells in fluorochrome-conjugated secondary antibody or fluorochrome-conjugated avidin, diluted in incubation
buffer at the recommended dilution.
8. Incubate for 30 min at room temperature.
9. Wash by centrifugation in 2 ml incubation buffer.
10. Resuspend cells in 0.5 ml 1X PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA
dye (Section E).
E. Optional DNA Dye
1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
2. Incubate for at least 30 min at room temperature.
3. Analyze cells in DNA staining solution on flow cytometer.
Flow Cytometry Alternate Protocol
(for combined staining of intracellular proteins and cell surface markers in blood)
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized or equivalent grade water.
1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH 2
O, mix.
2. 16% Formaldehyde (methanol free)
3. Triton X-100: To prepare 50 ml of 0.1% Triton X-100 add 50 µl Triton X-100 to 50 ml 1X PBS and mix well.
4. 50% methanol
5. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.
6. Secondary Antibodies: Anti-mouse (#4408, #8890, #4410, #8887), Anti-rabbit (#4412, #8889, #4414, #8885), Anti-rat
(#4416, #4418)
Fluorochrome
Reference Table
Max. excitation
wavelength (nm)
Max. emission
wavelength (nm)
Excitation
laser line (nm)
Color
Brilliant Violet 421 407 421 405 violet
DAPI 345 455 360/405 blue
Pacific Blue 404 456 360/407 blue
Hoechst 355 465 360 blue
Alexa Fluor ® 488 495 519 488 green
FITC 495 519 488 green
Pacific Orange 403 551 360/407 orange
Cy 3 548 561 488/532/561 orange
Alexa Fluor ® 555 555 565 488/532/561 orange
Phycoerythrin (PE) 496/566 576 488/532/561 orange
Texas Red ® 595 613 568/595 red
PE-Texas Red ® 496/566 616 488/532 red
Alexa Fluor ® 594 590 617 561/595 red
Propidium iodide (PI) 536 617 488/532 red
7-AAD 546 647 488/532 red
Allophycocyanin (APC) 650 660 633/635 red
Alexa Fluor ® 647 650 665 633/635 red
Cy 5 647 665 633/635 red
PE-Cy 5 496/546 666 488/532/561 red
PerCP 482 678 488/532 red
DRAQ7 599/644 678/694 635/647 red
DRAQ5 ® 647 681/697 488/633/647 red
PE-Cy 5.5 565 693 488 red
APC-Cy 5.5 650 694 633/635/647 red
PerCP-Cy ® 5.5 482 695 488 red
DyLight 680 692 712 680/685 red
Alexa Fluor ® 700 696 719 633/635 near-IR
APC-Cy 7 650 785 633/635 near-IR
PE-Cy 7 496/566 785 488/532/561 near-IR
DyLight 800 777 794 785 near-IR
Alexa Fluor ® 790 784 814 785 near-IR
10 0 0 200 400 600 800 1000
DNA (PI)
Propidium Iodide (PI)/RNase Staining
Solution #4087: Flow cytometric analysis
of Jurkat cells using Cyclin B1 (D5C10)
XP ® #12231 Rabbit mAb and #4087 (DNA
content). Anti-rabbit IgG (H+L), F(ab’) 2
Fragment (Alexa Fluor ® 488 Conjugate)
#4412 was used as a secondary antibody.
204 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.
www.cellsignal.com/cstprotocols
205