TITLE: Detection of phospho IRF3 and p65 in PBMCs by flow cytometry
AUTHORS: Andrea Bausch

[DESCRIPTION]
This protocol describes a method to detect phosphorylated IRF3 and phosphorylated p65 (subunit of NFkB) in PBMCs by flow cytometry. An extracellular staining to identify different immune subsets is followed by an intracellular staining steps for the detection of phospho proteins

[STEPS]
SECTION: Before start
1. Prepare DC Medium and pre-warm to 37 °C:   
+ 5% Human Serum 
+ 1%   
+ 1%  

Prepare Flow Buffer and cool to 4 °C: 
 
+2% heat inactivated FBS 
+ 2mM    

Put an aliquot of Methanol in the freezer and an aliquot of   and PBS in the fridge 
Cool centrifuge to 4 °C
SECTION: Preparation and stimulation of cells
2. Thaw PBMCs and adjust concentration to 1.11*10^7 cells/ml 

SECTION: Preparation and stimulation of cells
3. Seed 90 µL in 96-well round bottom plate (1x10^6 cells per well). 

SECTION: Preparation and stimulation of cells
4. Prepare a 10x stock solution of your stimulus. 



 


SECTION: Preparation and stimulation of cells
5. Add 10µl of your prepared stimulus to your cells 
SECTION: Preparation and stimulation of cells
6. Incubate at 37C, 5%CO2 for the desired time
 

SECTION: Extracellular Staining
7. All following steps have to be performed on ice using ice cold buffers. All centrifugation steps need to be performed at 4 °C 
Put an Aliquot of PBS, Flow Buffer and HistoFix on ice
SECTION: Extracellular Staining
8. Spin cells down 300 x g, 5 min, 4 °C 
Discard supernatant
Resuspend in 200µl Flow Buffer 
Spin cells down 300 x g, 5 min, 4 °C 
Discard supernatant

SECTION: Extracellular Staining
9. Prepare   and staining mixes. Keep in the fridge until use. 

SECTION: Extracellular Staining
9.1. Fc Block: Dilute 1:20 in Flow Buffer. Volume needed: 50 µL per sample

SECTION: Extracellular Staining
9.2. Master Mix: 
Prepare in BrilliantStainBuffer: 

Master Mix for 1 sample: 
            Fluorochrome  Markers  Dilution [1:…]  µl/test  µl Ab     BV421  CD3  50  1  1    BV510  CD14  20  2.5  2.5    BV605  HLA-DR  50  1  1    BV711  CD56  50  1  1    BV785  CD123  50  1  1    FITC  CD19  5  10  10    PE           0    PerCP/Cy5.5           0    PE-Cy7           0    AF647  CD11c  100  0.5  0.5    AF700           0    eFluor780  Via eF780  500  0.1  0.1          µl Brilliant Stain Buffer  32.9  
 


SECTION: Extracellular Staining
9.3. Prepare intracellular staining mix
Dilute pIRF3 or pP65 antibody 1:50 in Flow Buffer
Volume needed: 50µl per sample
SECTION: Extracellular Staining
9.4. Prepare staining mixes for controls
e.g. Viability Dye Single Staining, FMO controls 
SECTION: Extracellular Staining
10. Resuspend in 50 µL (1:20 pre-dilution in Flow Buffer)
Incubate 15 min on ice 
SECTION: Extracellular Staining
11. 300 x g, 5 min, 4 °C 
Discard supernatant

SECTION: Extracellular Staining
12. Resuspend cells in 50µl MasterMix 
Incubate for 20 minon ice in the dark (cover with aluminum foil to protect from light)

SECTION: Extracellular Staining
13. Add 150µl of Flow Buffer per well
Spin cells down 300 x g, 5 min, 4 °C 
Discard supernatant
Resuspend in 200µl Flow Buffer 
Spin cells down 300 x g, 5 min, 4 °C 
Discard supernatant

SECTION: Fixation and permeabilization
14. Resuspend cells in 100µl of FlowBuffer
Add 100µl of ice cold 4% HistoFix
Incubate on ice for 10 min in the dark

SECTION: Fixation and permeabilization
15. Spin cells down 500 x g, 5 min, 4 °C 
Discard supernatant
Resuspend in 200µl ice cold PBS
Spin cells down 500 x g, 5 min, 4 °C 
Discard supernatant
SECTION: Fixation and permeabilization
16. Resuspend cells slowly in 180µl ice cold Methanol
Incubate for 30 min - 60min at 4 °C in the fridge

SECTION: Fixation and permeabilization
17. Spin cells down 500 x g, 5 min, 4 °C 
Discard supernatant
Resuspend in 200µl ice cold FACS Buffer
Spin cells down 500 x g, 5 min, 4 °C 
Discard supernatant
Resuspend in 200µl ice cold FACS Buffer
Spin cells down 500 x g, 5 min, 4 °C 
Discard supernatant
SECTION: Intracellular Staining
18. Resuspend cells in 50µl intracellular staining mix
Incubate 30 min at 4C

SECTION: Intracellular Staining
19. Add 150µl of Flow Buffer per well
Spin cells down 500 x g, 5 min, 4 °C 
Discard supernatant
Resuspend in 200µl Flow Buffer 
Spin cells down 500 x g, 5 min, 4 °C 
Discard supernatant
SECTION: Intracellular Staining
20. Resuspend in 100µl of Flow Buffer and keep at 4C protected from light until acquisition