Homemade EU click

1 rxn = 1 single 24-well (250 µL EU or 175 µL click mix).

Note that ascorbic acid should be made fresh and EU will likely be the limiting reagent.

Warning

Click chemistry is known to diminish fluorescence due to ROS that are produced during the Cu-catalyzed azide-alkyne cycloaddition. Have not tested but the ThermoFisher Click-iT Plus EdU kit supposedly fixes this due to “the use of the picolyl azide combined with a copper protectant is the basis of the upgraded Click-iT Plus EdU technology, which achieves the same sensitive, reliable detection of cell proliferation as the original Click-iT EdU assay while also preserving the fluorescence of GFP, RFP.”

Important

The NaN3 is important to include in any single color controls! We observe that presence of EU doesn’t change CFSE only control signal but any azide addition (with or without EU) significantly changes FSC/SSC and CFSE signal. The following image shows the effects of azide addition on 1 dpi CFSE-labeled MEFs flowed at 4 dpi.

Stock solutions

• Main solutions

Stock solutions	Stock concentration	How to make	Final concentration
EU (100X)	100 mM	5 mg EU + 186 µL DMSO (74 rxns)	1 mM
CuSO4.5H2O (100X)	200 mM	250 mg CuSO4.5H2O + 5 mL H2O (2,800 rxns)	2 mM
Ascorbic acid (10X) MAKE FRESH	200 mg/ml	120 mg Ascorbic acid + 600 µL H2O (34 rxns)	20 mg/mL

• Main azide solutions

Stock solutions	Stock concentration	How to make	Final concentration
NaN3 (use to make 500X soln)	4 M	250 mg NaN3 + 961 µL H2O	–
NaN3 (500X)	4 mM	1 µL of 4 M NaN3 + 1,000 µL H2O (2,800 rxns)	8 µM
Sulfo-Cy5 azide (500X)	4 mM	5 mg Sulfo-Cy5 azide + 1,638 µL H2O (4,600 rxns)	8 µM
BDP TR azide (500X)	4 mM	5 mg BDP TR azide + 2,469 µL DMSO (7,000 rxns)	8 µM

• Misc/wash solutions

Stock solutions	How to make
0.5% Tween-20 in PBS	50 mL PBS + 250 µL Tween-20. Add Tween-20 to 1 mL PBS first to mix easier!
3.7% PFA in PBS	44 mL PBS + 5.7 mL 32% PFA
(Optional) 0.1 µg/mL DAPI in PBS	Dilute 0.1 mg/mL DAPI (1,000X stock in -20/Olaf) into PBS

Note

The 200 mg/mL ascorbic acid (10X) should be made fresh day of. It takes a while to dissolve so start it early and vortex often.

Warning

Azides are toxic! Always use azides in the fume hood and do not breathe it in! Azide waste should be collected in the satellite accumulation area in the fume hood.

Add EU to cells

1. In BSC hood, add 2.5 µL of 100 mM EU (100X) to 250 µL of complete growth medium (DMEM/FBS or N3 with no neurotrophics) per 24-well. For a full 24-well plate, 60 µL into 6 mL (90 µL into 9 mL if need more for single color controls).

2. Incubate cells for 1 hour at 37°C.

Dissociate

3. Remove media, wash with PBS, trypsinize, and spin down in eppendorfs.

Note

We did not notice a difference but you can precoat with 7.5% BSA/PBS and/or use low-retention eppendorfs. For 7.5% BSA/PBS precoating, just pipette up and down (no need to incubate).

Fix cells

4. Add 100 µL of 3.7% PFA/PBS to each Eppendorf and mix well. Let sit 15 at RT in the dark (covered).

5. Add 1 mL PBS and spin cold at 4°C to pellet cells.

6. Remove supernatant.

Permeabilize

7. Add 100 µL 0.5% Tween/PBS. Let sit 15 minutes at RT in the dark (covered).

8. Wash by adding 900 µL PBS and spin down at 4°C.

(Alt to step 8) Just add 175 µL of label mix to cells.

Note

Other protocols use 0.5% Triton X-100, but comparing Tween-20 and Triton X-100 we observed that the pellets permeabilized with Triton disappeared after the click step.

Important

Any immunostaining should be done at this point, before the click reaction. Staining after the click reaction results in high levels of non-specific staining and no detectable specific signals.

Prepare click/label mix

9. Make label mix just before use by combining (in this order: precipitate is formed after addition of CuSO4 to PBS, this is dissolved after addition of ascorbate):

   • The following mix is enough for a full 24-well plate: 4,500 µL ~ 25 rxns (OR 1,000 µL for NaN3 ctrls)

   Solution	Volume (for NaN3 ctrls)
   PBS	4,000 µL (or 888 µL)
   CuSO4 (100X, 200 mM)	45 µL (or 10 µL)
   Azide (500X, 4 mM)	9 µL (or 2 µL)
   Ascorbate (10X, 200 mg/mL)	450 µL (or 100 µL)

Label and wash

10. Add 175 µL click/label mix per tube and incubate cells 30 min at RT with label mix on rotator, protected from light, at room temp.

11. Wash by adding 900 µL PBS and spin down at 4°C.

12. (Optional) Incubate cells 5 minutes with 175 µL of 0.1 µg/mL DAPI/PBS if you want to detect nuclei in flow

13. Wash by adding 900 µL PBS and spin down at 4°C.

14. Analyze by flow.

Note

All spins are performed at ~500 rcf for 5 min. Our centrifuge follows RCF = 1e-4*[rpm]^2 + 4e-2*[rpm] - 6e1, where 2200 rpm = 512 rcf. It is recommended to perform all spins at 4°C once the cells have been fixed to prevent pellet loss.