TITLE: U54 SCENT Transcription Factor (TF)-Phosflow Flow Cytometry Panel
AUTHORS: Zachary R Healy, david murdoch, alicia cooper-volkheimer

[DESCRIPTION]
This flow cytometry panel was developed for simultaneous quantification of transcription factors, phosphorylated kinases, and endosomal β-galactosidase activity in human peripheral blood mononuclear cell (PBMC) subsets including T cells, B-cells, NK cells, and monocytes. This protocol begins immediately following cell thawing, counting, viability determination, and peptide stimulation. This is detailed in SENNET Antibody Staining Protocol. 







[STEPS]
SECTION: General Notes
1. Instrument QC, Single-Stain Controls, Fluorescence minus-one (FMO) controls, and General Protocol Notes . We routinely perform single-stain controls (using both cells and compensation beads) as well as FMO for the intracellular targets (highlighted in BOLD in materials section) prior to any batch processing.
SECTION: General Notes
1.1. Instrument QC. The instrument, a Cytek Aurora NorthernLights, 3-laser (405nm, 488nm, 605 nm) undergoes daily QC prior to any experimentation. Experiments are only performed after the instrument passes QC. This is performed after a 30-minute laser warm-up period using 1 drop of Cytek SpectroFlow QC beads (current lot 2003) in 200 uL of DI water. All QC reports are maintained on a local server and the desktop hard-drive for monitoring. The instrument receives annual preventive maintenance is under an active service contract with Cytek. 
SECTION: General Notes
1.2. Single-Stain Controls. Single stain controls are obtained using both cells (from a known batch control normal/healthy donor maintained in our laboratory repository following leukapheresis) and using BD compensation beads. All single stain controls undergo the entire protocol prior to acquisition, including all incubations listed below for the designated durations to ensure a reliable representation each step has on the fluorophore. 
SECTION: Stimulation with CEF Peptide
2. Following thawing, viability assay, counting, and distribution of cells to the 96-well plate, stimulation using CEF extended pool I (MHC I) and CEFX Ultrastim Pool (MHC II) is performed as detailed in SENNET Antibody Staining Protocol using a final concentration of each peptide at 1.0 µg/µL in a 225 µL total volume with 2 x106 cells/well starting concentration. The DMSO concentration is maintained at a maximum of 1% during this process, and DMSO solvent control is used for non stimulated cells as an equivalent concentration. The stimulation is performed for 6 hours after which the cells are transferred to a 4C refrigerator until surface staining is begun. 
SECTION: General Notes
1.3. FMO controls. FMO controls are performed for each of the intracellular targets above prior to any batch processing of cells; however, due to the nature of targets in this panel, we do use isotype controls during the FMO experiments as recommended by the majority of the manufacturers above. 
SECTION: General Notes
1.4. General Protocol Notes. First, we use BD Brilliant Stain Buffer Plus in all surface and intracellular staining steps of the protocol at 10 µL per 100 µL staining volume. As all our staining is performed at 50 µL, we use 5 µL per test. In addition, during surface staining, we use Biolegend TruStain FCX blocking agent at 5 µL per 100 µL volume (2.5µL in 50 µL). All stimulation and staining is performed in Costar 96-well U-bottom TC-treated PS plates at a starting concentration of 2 x 106 cells per well. Stimulations with overlapping peptides are performed for 6 hours at 37C in a 5% CO2, humidified incubator environment. All centrifugation steps are performed in an Eppendorf 5430 swinging bucket centrifuge at room temperature (centrifugation speeds vary by step, please be aware). All experiments use a batch control.
None. 
SECTION: Surface Staining
3. Prepare surface staining cocktail using the following table in the indicated order, adding 10% of each reagent to account for pipetting errors (see table in 3.1)


SECTION: Surface Staining
3.1. 
     Surface Stain      Amount per Test        Cell Staining Buffer (base panel)      33.64        BD Brillant Stain Buffer Plus      5        TruFCX block      2.5        (mix by pipetting)               ZnIR      0.1        CD3 APC-Fire810      1.25        CD14 BV570      2.5        CD19 BV605      0.63        CD56 BV510      1.25        CD39 R718      0.63        CX3CR1 BV711      2.5   

SECTION: Surface Staining
3.2. Centrifuge the cells in the 96-well plate at 300xg for 3 minute (wash 1)
SECTION: Surface Staining
3.3. Decant supernatant, being careful not to disrupt cell pellet. 
SECTION: Surface Staining
3.4. Wash the cells in 200 µL of cell staining buffer (wash 2). 
SECTION: Surface Staining
3.5. Centrifuge the cells in the 96-well plate at 300xg for 3 minutes.
SECTION: Surface Staining
3.6. Decant supernatant, being careful not to disrupt cell pellet. 
SECTION: Surface Staining
3.7. Place the cells in an Eppendorf MixMate to release the cell pellet for 30s at 600 rpm. 
SECTION: Surface Staining
3.8. Pipette 50 µL of surface staining cocktail to each well. 
SECTION: Surface Staining
3.9. Cover with aluminum foil and incubate at 4C for 30 minutes. 
SECTION: Surface Staining
3.10. During this incubation, we typically prepare the reagents for the next step of the protocol (β-galactosidase activity assay). 
SECTION: Surface Staining
3.11. Following incubation, add 150 µL of cell staining buffer to each well (wash 1). 
SECTION: Surface Staining
3.12. Centrifuge the cells in the 96-well plate at 300xg for 3 minute.
SECTION: Surface Staining
3.13. Decant supernatant, being careful not to disrupt cell pellet. 
SECTION: Surface Staining
3.14. Wash the cells in 200 µL of cell staining buffer (wash 2).   
SECTION: Surface Staining
3.15. Centrifuge the cells in the 96-well plate at 300xg for 3 minutes.
SECTION: Surface Staining
3.16. Decant supernatant, being careful not to disrupt cell pellet. 
SECTION: Surface Staining
3.17. Wash the cells in 200 µL of cell staining buffer (wash 3). 
SECTION: Surface Staining
3.18. Centrifuge the cells in the 96-well plate at 300xg for 3 minutes.
SECTION: Surface Staining
3.19. Decant the supernatant, being careful not the disrupt the cell pellet. 
SECTION: Surface Staining
3.20. Place the cells in an Eppendorf MixMate to release the cell pellet for 30s at 600 rpm. 
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4. Optimal β-galactosidase staining conditions were determined using a single-color high-throughput approach in human primary T cells with Palbociclib incubation (5 µM) in the presence of anti CD2/anti-CD3/anti-CD28 (StemCell) and high-dose IL-2 (500 IU/mL; Miltenyi IL-2 IS) for 7 days to induce β-galactosidase expression. The optimal conditions were 10 minute fixation at 0.5X BL Fixative solution, followed by 90 minute incubation at a concentration of 1:750 of the β-galactosidase Green Probe.
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.1. Resuspend the cell pellet in 100 µL of 0.5X Biolegend Fixative Solution (1:1 D-PBS w/o Mg, Ca and 1x Fixative solution).    

SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.2. Incubate the cells for 10 minutes at room temperature in the dark. 
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.3. Following incubation, add 100 µL of cell staining buffer to each well (wash 1).
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.4. Centrifuge the cells in the 96-well plate at 300xg for 3 minutes.
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.5. Decant supernatant, being careful not to disrupt cell pellet. 
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.6. Wash the cells in 200 µL of cell staining buffer (wash 2). 
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.7. Centrifuge the cells in the 96-well plate at 300xg for 3 minutes.
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.8. While cells are centrifuging, prepare β-galactosidase Working Solution using the following table, including 10% extra of each component to account for pipetting errors/variability:

 
 
  
  Component per test (1:750 dilution)
  
  
  
  βgal Green Probe
  
  
  0.133 µL/test
  
  
  
  Senescence Diluent Buffer
  
  
  100 µL/test
  
    
 
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.9. Incubate the cells in 100 µL of Working Solution for 90 minutes at 37C without CO2 in the dark. 
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.10. Following incubation, add 100 µL of cell staining buffer to each well (wash 1). 
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.11. Centrifuge the cells in the 96-well plate at 300xg for 3 minutes.
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.12. Decant supernatant, being careful not to disrupt cell pellet. 

SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.13. Wash the cells in 200 µL of cell staining buffer (wash 2). 
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.14. Centrifuge the cells in the 96-well plate at 300xg for 3 minutes.
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.15. Decant supernatant, being careful not to disrupt cell pellet. 
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.16. Wash the cells in 200 µL of cell staining buffer (wash 3). 
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.17. Centrifuge the cells in the 96-well plate at 300xg for 3 minutes.
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.18. Decant supernatant, being careful not to disrupt cell pellet. 
SECTION: β-galactosidase activity assay (Cell Event Senescence Green Kit)
4.19. Place the cells in an Eppendorf MixMate to release the cell pellet for 30s at 600 rpm. 
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5. First, it should be noted that the fixation and permeabilization conditions vary for cytokines, transcription factors, and phosphorylated targets. While mild permeabilization is necessary for cytokine staining in cytoplasm, harsher conditions are necessary for staining nuclear targets. In particular, methanol permeabilization is recommended for some transcription factors and phosphorylated targets. The downside to the use of methanol is that methanol is detrimental to many fluorophores (particularly protein-based and tandem dyes) and alters epitopes of may proteins. Many transcription factors and phosphorylated kinases can be successfully stained using paraformaldehyde fixation followed by more harsh permeabilization (typically involving triton-X or higher saponin concentrations than typically used in cytokine staining). This approach has the advantage of improved signal-to-noise ratio/reduced background staining and less negative effect on fluorophores. We tested the BL TruePhos (methanol buffer), the BL True Nuclear (detergent-based), and the BD PhosFlow buffer III (methanol) for optimal performance. Overall, the BL True Nuclear buffer provided the best performance overall. Although the staining of p16 was less optimal (although still able to be resolved), the staining of NFkB, p38, p21, and H2Ax was improved compared to the methanol-based fixative. In addition, this allowed for the inclusion of addition cell surface staining targets (CD39, CX3CXR1) which we would not have been able to include otherwise.  
      
This protocol is based on the recommendations of Biolegend for the use of this kit in 96-well plate format. Please refer to the following for further details (https://www.biolegend.com/protocols/true-nuclear-transcription-factor-staining-protocol-for-96-well-u-bottom-plate/4246/). Note that the prior fixation performed during the β-galactosidase assay (section 4) was mild and it was necessary during optimization to further fix the cells after the β-galactosidase assay to improve background staining. 
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.1. Prepare 1x Biolegend TrueNuclear Fix Concentrate by mixing 1-part 4X Fix Concentrate with 3 parts Fix Diluent to allow for 200 µL of 1X Fix Concentrate per well (e.g., for 10 samples, one would need 2200 µL (including 10% additional) of 1x Fix Concentrate; therefore, this would require 550 µL of 4x Fix and 1650 µL of 4x Fix Diluent). 
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.2. Resuspend the cell pellet in 200 µL of 1x Fix Concentrate for 60 minutes at RT in the dark.
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.3. Following incubation, centrifuge the cells in the 96-well plate at 500xg. Note the increased speed which will be necessary to pellet the cells following permeabilization.
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.4. Decant supernatant, being careful not to disrupt cell pellet

SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.5. Wash the cells in 200 µL of 1x PermWash (wash 1). 
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.6. Centrifuge the cells in the 96-well plate at 500xg for 3 minutes.
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.7. Decant supernatant, being careful not to disrupt cell pellet
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.8. Wash the cells in 200 µL of 1x PermWash (wash 2). 
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.9. Centrifuge the cells in the 96-well plate at 500xg for 3 minutes.
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.10. Decant supernatant, being careful not to disrupt cell pellet
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.11. Wash the cells in 200 µL of 1x PermWash (wash 3). 
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.12. Centrifuge the cells in the 96-well plate at 500xg for 3 minutes.
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.13. Place the cells in an Eppendorf MixMate to release the cell pellet for 30s at 600 rpm. 
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.14. Prepare the Intracellular Staining Mix/Cocktail using the following table, in the order listed:

 
  
  ICS Panel
  
  
  Amount Per Test
  
  
  
  1xPermWash
  
  
  35.5
  
  
  
  BD Brilliant Stain Buffer Plus
  
  
  5
  
  
  
  phospho-gH2Ax (Ser139) BV421
  
  
  2.5
  
  
  
  Bcl-2 PE-CF594
  
  
  2.5
  
  
  
  phospho-NFkB (Ser529) PerCP-efluor710
  
  
  2.5
  
  
  
  phospho-p38 (Thr180, Tyr182) PE-Cy7
  
  
  2.5
  
  
  
  p16 PE
  
  
  2
  
  
  
  p21 AF647
  
  
  2.5
  
    
 
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.15. Resuspend the cell pellets in 50 µL of the Intracellular Staining Mix. 
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.16. Incubate at RT for 45 minutes in the dark. 
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.17. Add 150 µL of 1x PermWash to each well (wash 1).
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.18. Centrifuge the cells in the 96-well plate at 500xg for 3 minutes.
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.19. Decant supernatant, being careful not to disrupt cell pellet. 
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.20. Wash the cells in 200 µL of 1x PermWash (wash 2). 
SECTION: Intracellular Staining for TF, Phosphorylated kinase targets using Biolegend TrueNuclear Kit.
5.21. Centrifuge the cells in the 96-well plate at 500xg for 3 minutes.
5.22. Decant supernatant, being careful not to disrupt cell pellet.   
5.23. Wash the cells in 200 µL of 1x PermWash (wash 3). 
5.24. Centrifuge the cells in the 96-well plate at 500xg for 3 minutes.
5.25. Place the cells in an Eppendorf MixMate to release the cell pellet for 30s at 600 rpm. 
5.26. Resuspend the cells in 200 µL of BL FluorFix Buffer and acquire the samples on the flow cytometer.