實驗概要
The measurement of cell proliferation is fundamental to the assessment of cell health, genotoxicity, and drug efficacy. Proliferation is traditionally assessed by incubating cells with a single “pulse” of a nucleoside analog that is incorporated into DNA and detected using radioactivity, antibodies, or click chemistry. Some applications, such as drug efficacy testing, benefit from the incorporation of two different analogs at different time points (dual pulse labeling), which can further define cell cycle kinetics. With the availability of new highly specific anti-BrdU antibodies, the BrdU labeling technique can be combined with click chemistry detection for a simplified method of dual pulse labeling.
Traditionally, the detection of cell proliferation has employed the incorporation of the thymidine analog BrdU (5-bromo-2′-deoxyuridine) during DNA synthesis, followed by detection with an anti-BrdU antibody. This method is rapidly being replaced by the click chemistry–based Click-iT? EdU assay, because unlike BrdU assays, Click-iT? EdU assays are not antibody-based and therefore do not require DNA denaturation for detection of the incorporated nucleoside. Click-iT? assays use a modified nucleoside, EdU (5-ethynyl-2′-deoxyuridine), that is incorporated during DNA synthesis and detected using a click reaction—a copper(I)-catalyzed reaction between an azide and an alkyne.
實驗材料
Materials
Jurkat cells in complete RPMI media/10%FBS
BrdU (5-bromodeoxyuridin) (B23151) (10mM in DMSO)
EdU (5-Ethynyl-2’-deoxyuridine) (10mM in DMSO)
PBS pH7.2 (GIBCO(R))
70% Ethanol, ice cold
4M HCl
Phosphate/citric acid buffer, pH 7.4 (182 ml of 0.2M Na2HPO4 18ml 0.1M citric acid)
Antibody Diluting buffer (PBS 0.1% Triton-X100 1% BSA)—prepare fresh
Purified anti-BrdU antibody(clone MoBU-1) (B35141) 0.1 mg/ml; use 2.5ul
anti-BrdU- (clone Br3) pure Caltag MD5300 lot 0903 0.4mg/ml titer use: 2.5uL
Goat anti-mouse IgG Alexa Fluor? 488 secondary antibody 2mg/ml, dilute 1:20 and use 2.5ul/test (0.25ug/test) (A11001)
Tris Buffered Saline
CuSo4 100mM
100mM ascorbate (Freshly prepared)
AF647-azide 20uM DMSO A10277
Note: (Instead of listing above AF647-azide and EdU separately, can we just say Click-iT EdU Alexa Fluor(R) 647 Flow Cytometry Assay (A10202) to promote OUR sku)
FxCycle? Violet 1mg/ml DI F10347 use 0.5μl/0.5ml
實驗步驟
1. Use Jurkat cells in complete RPMI/10%FBS media, separate into 5 flasks
2. Flask 1: control; flask 2: 10uM BrdU 1 hour; flask 3: 20uM EdU 1 hour; flask 4: 10μM BrdU 20μM Edu EdU together 1 hour; flask 5: 20μM EdU one hour then 10μM BrdU one hour. incubate 37°C/5%
3. After incubation, Centrifuge cells, pellet and remove supernatant.
4. FIX cells: Add 10ml ice cold 70% Ethanol to a 15ml tube containing the cell pellet, adding dropwise at first while vortexing, mix well.
5. Store at -20°C until use, 4 hours to several months.
6. Centrifuge cells, pellet & discard supernatant ; vortex pellet
7. Resuspend pellet in 2.0ml 4M HCl for 20 min at room temperature.
8. Add 10ml phosphate/citric acid buffer, centrifuge and decant supernatant. Filter out large clumps
9. Reusupend in PBS/0.1%Triton/1%BSA (antibody stain solution) wash & pellet.
10. Repeat wash.
11. Reusupend in PBS/0.1%Triton/1%BSA (antibody stain solution), adjust concentration to 1x107/ml.
12. Add 100μl cell suspension to designated tubes.
13. Prepare click reaction and add 0.5ml to designated tubes
14. Incubate 30 minutes at room temperature protected from light
15. Wash x1 PBS/0.1%Triton/1%BSA
16. Add anti-BrdU purified antibody to designated tubes
17. Incubate for 30 minutes at room temperature protected from light
18. Wash with 3ml PBS/0.1%Triton/1%BSA (antibody stain solution) spin & pellet.
19. Add Goat anti-mouse IgG Alexa Fluor? 488 secondary antibody, incubate for 30 minutes at room temperature, followed by wash as in step 15.
20. Resuspend pellet in 0.5ml PBS/0.1%Triton/1%BSA (antibody stain solution)
21. Add 0.5μL FxCycle? Violet Stain
22. Incubate for 15-30 minute at room temperature protected from light
23. Run on LSRII
24. Analyze Alexa Fluor? 488 using blue laser excitation and 525/50 emission filter, Alexa Fluor? 647 using red laser excitation and 660/20 emission filter, and FxCycleViolet using violet laser excitation and 450/50 emission filter,
25. Gate on main population cells and collect 10,000 gated events for single color data. For triple color samples, make a singlet gate on WvA and collect 20,000 singlet events.