This note provides a detailed protocol for flow cytometry-based screening of a given target’s phosphorylation level using one of Abwiz Bio’s many phosphorylation-specific rabbit monoclonal antibodies. This protocol has been rigorously optimized and is used by our own scientists for product validation. If you have any questions about this protocol or need help troubleshooting your own experiments, our Ph.D.-level support team is standing by to help you publish great results!

1. Cells, cultured to >90% viability and 80-90% confluency
2. T75 cell culture flask (or appropriate cell culture vessel)
3. PBS
4. 0.5% trypsin solution
5. 50 mL falcon tubes
6. 16% paraformaldehyde solution
7. Ice-cold 100% methanol
8. 96-well V-bottom plate
9. FACS buffer (1% BSA, PBS, 0.05% sodium azide)
10. Phospho-specific IgG antibody (choose from Abwiz Bio’s large  collection of validated mAbs!)
11. Isotype control antibody (e.g. Abwiz Bio Cat. #2141)
12. Anti-rabbit IgG secondary antibody
13. Flow cytometer with 96-well plate reader

The cell treatment procedure will vary depending on the cell line and the desired treatment(s) to be used. This representative protocol is for HeLa (adherent) cells.

1. Cell culturing. Culture the desired cell line in the appropriate medium to>90% viability. Seed cells the day before in T75 flasks. When cells reach 80-90% confluence, aspirate media to remove.
2. Cell treatment. Add the appropriate medium containing the desired amount of treatment
   additive. Incubate cells at 37°C for the desired length of time.
   a. Treatment conditions will vary based on the phospho target and cell type and may
   require optimization.
3.   Detach cells. Aspirate the media supernatant. Wash cells by adding 10 mL of room-temperature PBS to the flask, then aspirate the PBS. Repeat for a total of two PBS washes. Add 1.5 mL of 0.5% trypsin and incubate at 37°C for 1-3 min. Detach cells from the flask by adding 10 mL of PBS and using gentle pipetting. Transfer cell suspension to 50 mL falcon tubes.
4. Wash cells. Pellet cells 1,500xg for 5min. Aspirate the supernatant and resuspend in 10 mL of PBS. Repeat for a total of two washes. Resuspend the final cell pellet in 2mL of PBS and count the cells.
5. Fix cells. Add 100 µL of 16% paraformaldehyde per 1 mL of cell suspension. Incubate cells at room temperature for 10 min.
6. Resuspend cells. Pellet cells 1,500 x g for 5 min. Aspirate most of the supernatant, resuspending the cell pellet in the remaining 500 µL – 1 mL solution. Add ice-cold 100% methanol to achieve 2-4x106 cells/mL density (and >90% final methanol concentration) and vortex to mix. Incubate cells for 20 min at 4°C. Cells can be used immediately for flow analysis or stored at -20°C for up to four months.

1. Cell aliquot and wash. Aliquot ~1x105 cells/well (25µL of 4x106 cells/mL suspension) to a 96-well V-bottom plate. Add 200 µL of FACS buffer to each well. Pellet cells 2,000 rpm for 5 min at 4°C. Aspirate supernatant.
2. Primary antibody incubation. Prepare primary solutions by diluting antibodies in FACS buffer. Resuspend cells in 100 µL of antibody solution by pipetting. Incubate at 4°C for 1 hour.
   1. The recommended dilution for each of Abwiz Bio’s phospho-specific IgG products isposted in the figure legend on the product page. Your experiment may requireoptimization by titration at multiple concentrations.
   2. Don’t forget to include the following controls:
      1. Secondary only – Resuspend cells in FACS buffer only during the primary incubation step
      2. No secondary – Resuspend cells in FACS buffer only during both the primary andsecondary incubation steps
      3. Isotype control – test at the same concentration as the primary antibody. For best results, use  Abwiz Bio isotype control rabbit mAb Cat. #2141.
3. Wash.Pellet cells 2,000 rpm for 5 min at 4°C. Aspirate supernatant. Wash by resuspending cells in 200 µL FACS buffer by pipetting. Repeat spin and aspirate supernatant.
4. Secondary antibody incubation.Prepare the secondary solution using an anti-rabbit IgG secondary antibody at the dilution recommended by the manufacturer. It may be necessary to titrate the secondary antibody to determine the optimal concentration. Resuspend cells in 100 µL of secondary antibody solution by pipetting gently. Incubate at 4°C for 30 min protected from light.
   1. All Abwiz Bio phospho-specific rabbit mAbs have been tested using DyLight™ 488 Donkey anti-rabbit IgG (Biolegend 406404) secondary antibody at 1:500 dilution.
5. Wash.Pellet cells 2,000 rpm for 5 min at 4°C. Aspirate supernatant. Wash by resuspending cells in 200 µL FACS buffer by pipetting. Repeat spin and aspirate supernatant.
6. Resuspend cells and read plate. Resuspend cells in 100 µL FACS buffer by pipetting. Read cells on flow cytometer.

Below are representative results from a titration study using  Phospho-Slp76 (Y128) rabbit  mAb clone Slp76Y128-3F8, Cat. #2136. The antibody was titrated from 1 µg/mL to 1 ng/mL using Ramos cells, serum starved overnight, then untreated or treated using 1mM pervanadate.