Click-&-Go™ EdU 594 Flow Cytometry Kit is optimized for imaging alkyne-tagged biopolymers with red-fluorescent AFDye 594 Azide (Alexa Fluor®594 Azide equivalent) using standard flow cytometry methods. The kit contains all components needed to detect the incorporated alkynes, including aldehyde-based fixation and detergent permeabilization reagents. Sufficient amount of reagents provided for 50 assays based on the protocol provided.
|AFDye 594 Azide (Component A)||—||—|
|Fixative (Component B)||4% PBS solution||5 mL|
|Copper (II) Sulfate (Component C)||100 mM||0.55 mL|
|Reducing Agent (Component D)||NA||400 mg|
|Saponin-based permeabilization and wash reagent (Component E)||10 x||50 mL|
Materials Required but Not Provided
- 1% BSA in PBS (pH 7.4)
- Flow cytometry tubes
- PBS buffer, pH 7.4
- Deionized water
- Fixative (Component B) contains paraformaldehyde, which is harmful. Use with appropriate precautions.
- Saponin-based permeabilization and wash reagent (Component E) contains sodium azide, which yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
|AFDye 594 Azide (Component A)||Add 140 µL of DMSO or water. This solution is stable up to 1 year if stored at –20°C protected from light.|
|1X saponin-based permeabilization and wash reagent (Component E)||To prepare 500 mL of 1X saponin-based permeabilization and wash reagent, add 50 mL of Component E to 450 mL of 1% BSA in PBS. Smaller amounts can be prepared by diluting a volume of Component E 1:10 with 1% BSA in PBS. After use, store any remaining solutions at 2–8°C. When stored as directed, the 1X solution is stable for 6 months and the 10X solution is stable for 12 months after receipt.|
|Reducing Agent (Component D)||Prepare 1 x solution of Reducing Agent (Component D) that is enough for one day. Weight our 20 mg of Reducing Agent (Component D) into 2 mL vial, add 1 mL of deionized water. Vortex until completely dissolved.
Note: reducing agent is susceptible to oxidation and turns brown when oxidized. If solution appears brown do not use.
Please visit Technical Resources section of our website to download a protocol for labeling cells with alkyne-containing metabolic labeling reagents.
1. Optional cell-surface antigen staining with antibodies
- After alkyne labeling wash cells once with 3 mL of 1% BSA in PBS, pellet cells by centrifugation, and remove supernatant.
- Dislodge the pellet and resuspend cells at 1 × 107 cells/mL in 1% BSA in PBS.
- Add surface antibodies, mix well and incubate for the recommended time and temperature protected from light.
- Proceed to cell fixation step.
2. Cell fixation and permeabilization
The following protocol is provided for fixation step using saponin-based permeabilization and wash reagent. This reagent can be used with whole blood or cell suspensions containing red blood cells, as well as with cell suspensions containing more than one cell type. This permeabilization and wash reagent maintains the morphological light scatter characteristics of leukocytes while lysing red blood cells. Protocols using other fixation/permeabilization reagents, such as methanol and 3.7% formaldehyde in PBS followed by a 0.5% Triton®X-100 permeabilization step also can be used.
- Wash the cells once with 3 mL of 1% BSA in PBS, pellet the cells, and remove the supernatant.
- Dislodge the pellet, add 100 μL of Fixative (Component B) and mix well, and incubate for 15 minutes.
- Remove the fixative and wash the cells in each with 1 mL of 1% BSA in PBS. If red blood cells or hemoglobin are present in the sample repeat the wash step. If require remove all residual cell debris.
- Remove the wash solution and resuspend the cells in 100 μL of 1X saponin-based permeabilization and wash reagent, and mix well. Incubate the cells for 15 minutes.
- Proceed directly to alkyne detection step or to treating cells with antibodies to intracellular antigens followed by EdU detection step.
3. Alkyne detection
Note: 500 μL is used per reaction. A smaller volume can be used as long as the remaining reaction components are maintained at the same ratios.
- Weight our 20 mg of Reducing Agent (Component D) into 2 mL vial, add 1 mL of deionized water, vortex until completely dissolved. This solution should be used freshly prepared on the same day.
- Prepare required amount of the reaction cocktail according to Table 1. Add the ingredients in the order listed in the table. Use the reaction cocktail within 15 minutes of preparation.
Reaction component Number of Reactions 1 2 5 10 15 50 PBS buffer 438 µL 875 µL 2.19 mL 4.38 mL 6.57 mL 21.9 mL Copper + Protectant (Component C) 10 µL 20 µL 50 µL 100 µL 150 µL 500 µL AFDye 594 Azide solution (Material preparation) 2.5 µL 5 µL 12.5 µL 25 µL 37.5 µL 125 µL 1x Reducing Agent (step 3.1) 50 µL 100 µL 250 µL 500 µL 750 µL 2.5 µL Total Volume 500 µL 1 mL 2.5 mL 5 mL 7.5 mL 25 mL
- Remove the permeabilization buffer (step 2.5).
- Add 0.5 mL of the Reaction Cocktail to each tube. Rock the tube briefly to insure that the reaction cocktail is distributed evenly.
- Protect from light, and incubate the tube for 30 minutes at room temperature.
- Remove the reaction cocktail.
Wash the cells well once with 3 mL of 1X saponin-based permeabilization and wash reagent.
Pallet the cells and remove the wash solution.
- If intracellular antibody labeling required dislodge the cell pellet and resuspend the cells in 100 μL of 1X saponin-based permeabilization and wash reagent.
- If no intracellular antibody labeling required add 500 μL of 1X saponin-based permeabilization and wash reagent.
At this point the samples are ready for DNA staining. If no DNA staining is desired, proceed to analyzing the cells on a flow cytometer.
4. Optional intracellular or surface antigens staining
- Add antibodies against intracellular antigens or against surface antigens that use RPE, PR-tandem or Quantum Dot antibody conjugates. Mix well.
- Protect from light and incubate the tubes for the time and temperature required for antibody staining.
- If Alkyne Detection has not yet performed, wash the cells once with 3 mL of 1% BSA in PBS, pellet the cells, and remove the supernatant. Dislodge the cell pellet and add 100 μL of 1x saponin-based permeabilization and wash reagent, mix well and proceed to Alkyne Detection (step 3.1)
- If Alkyne Detection has been performed (steps 3.1–3.8), wash each tube with 3 mL of 1X saponin-based permeabilization and wash reagent, pellet the cells, and remove the supernatant. Dislodge the cell pellet and resuspend the cells in 500 μL of 1X saponin-based permeabilization and wash reagent, and proceed to DNA staining, or to the cell analysis on a flow cytometer.
5. Optional DNA staining
- Add the appropriate DNA stain to each tube and mix well.
- Protect from light and incubate as recommended by a manufacture.
- Remove the DNA stain and wash each tube with 3 mL of 1X saponin-based permeabilization and wash reagent each well.
Flow Cytometry Analysis
Use a low flow rate during acquisition, if a traditional flow cytometer with a hydrodynamic focusing is used to measure the total DNA content. The same collection rate and cell concentration should be used for each sample within an experiment. Detect the fluorescent signal generated by DNA content stains with linear amplification. The fluorescent signal generated by labeling is best detected with logarithmic amplification.
The excitation and emission maxima of AFDye 594 are 590 nm and 617 nm.