TITLE: U54 SCENT Intracellular Staining (ICS) Senescence Flow Cytometry Panel
AUTHORS: Zachary R Healy, David Murdoch, Alicia Cooper-Volkheimer

[DESCRIPTION]
This protocol describes the Intracellular Staining (ICS) Senescence Flow Cytometry Panel


[STEPS]
SECTION: FBS Aliquot Prep
1. FBS (hiFBS): Gemini Bio Products Cat #100-106 1L
Prepare FBS Aliquots:
SECTION: FBS Aliquot Prep
1.1. Thaw heat-inactivated FBS (hiFBS):
Thaw a 500mL Bottle of FBS at 4°C. This may require more than overnight so the 500mL Bottle may be removed 2 or 3 days prior to use. Do not leave the FBS at room temperature overnight.
SECTION: FBS Aliquot Prep
1.2. Aliquot the 500mL FBS Bottle in 50mL aliquots (a total of ten 50mL hi-FBS Aliquots)
SECTION: FBS Aliquot Prep
1.3. Label the aliquots with Batch#, Expiration Date, Aliquot Date
SECTION: FBS Aliquot Prep
1.4. Store 50mL aliquots @ -20ºC until expiration date or for up to 2 freeze/thaw cycles
SECTION: FACS Wash
2. FACS Wash w/ EDTA (D-PBS with 0.5% FBS + 2 mM EDTA): Invitrogen Cat # 41190-250
SECTION: FACS Wash
2.1. Remove 4.5mL PBS from a 500mL bottle
SECTION: FACS Wash
2.2. Add 2.5mL of thawed FBS
SECTION: FACS Wash
2.3. Add 2mL of 0.5M EDTA solution (Catalog #: E7889-100ML)
SECTION: FACS Wash
2.4. Label the bottle with preparer's initials and expiration date (one month from preparation)
SECTION: FACS Wash
2.5. Store @ 4°C
SECTION: Pen-Strep-Glut (PSG)
3. Pen-Strep-Glut (PSG) (L-Glutamine-Penicillin-Streptomycin Soln): Sigma Cat#: G6784-100ML
SECTION: Pen-Strep-Glut (PSG)
3.1. Thaw 100mL bottle
SECTION: Pen-Strep-Glut (PSG)
3.2. Aliquot into 10mL into 15mL conicals (total of ten 15mL conicals)
SECTION: Pen-Strep-Glut (PSG)
3.3. Label the aliquots with Batch#, Expiration Date, Aliquot Date
SECTION: Pen-Strep-Glut (PSG)
3.4.  Store @ -20°C
SECTION: R10FBS Media Preparation
4. R10FBS Media Preparation (“R10”)
SECTION: R10FBS Media Preparation
4.1. Remove 55mL RPMI from a 500mL bottle of RPMI
SECTION: R10FBS Media Preparation
4.2. Add 50mL aliquot of thawed, hiFBS
SECTION: R10FBS Media Preparation
4.3. Add 5mL aliquot of thawed Pen-Strep-Glut
SECTION: R10FBS Media Preparation
4.4. Label the bottle with preparer's initials and expiration date (one month from preparation)
SECTION: Cytofix/CytopermSolution (10X) with GolgiStop (monensin)
5. Cytofix/Cytoperm Solution (10X) with GolgiStop (monensin): BD Biosciences Cat # 554715

Kit Contents :             
125mL 10X Perm/Wash Buffer 
100mL Cytofix Solution
0.7mL GolgiStop (monensin)

To prepare 1X Perm Wash:
1. Dilute 10X BD Perm/Wash buffer in distilled H2O to make a 1X solution prior to use
SECTION: Formaldehyde Solution
6. 1% Formaldehyde Solution (“1% Fix”) (“PFA”)

Reagents:    
10% Formalin
PBS
SECTION: Formaldehyde Solution
6.1. Add 5 mL 10% Formalin to a sterile 50 mL centrifuge conical tube
SECTION: Formaldehyde Solution
6.2. Add 45 mL PBS
SECTION: Formaldehyde Solution
6.3. Label the bottle with reagent name, initials & expiration date (one month from preparation)
SECTION: Formaldehyde Solution
6.4. Store 1% Fix at Room Temperature (18-25°C) for up 1 month

SECTION: PepMixes
7. PepMixes:

PepMix CEFX Ultra SuperStim Pool MHC-II Subset, JPT Technologies Cat #:PM-CEFX-3
PepMix CEF Pool (extended), JPT Technologies Cat #: PM-CEF-E-1
SECTION: PepMixes
7.1. Resuspend peptide pellet [25ug] in 50uL of DMSO [500ug/mL]
                
Add 10uL DMSO at a time, with vortexing to resuspend



SECTION: PepMixes
7.2. Aliquot and store at -20°C until day of use
SECTION: PepMixes
7.3. On day of use, use 0.45uL per test



SECTION: Protocol
8. Overview

Day 1: Thaw cells and distribute to 2 plates. Rest plates at 37C/5% CO2 for >6 hours
Overnight: Stim for 6 hours
Day 2: 1 Stim + 1 Unstim plate stain for surface and ICS then acquire flow data
Time experiment steps according to lab and anticipated acquisition schedule
SECTION: Protocol
8.1. Day 1 – Thawing
1.  Prepare 20ml of R10 in 50mL conical tube per sample (1-4 vials per tube)

2. Warm the R10 for 30 min at 37C prior to use

3. Place cryovials in a 37C bath for 3-5 sec at a time. Withdraw, examine and repeat (usually 3-4 rounds) until small, pea-sized amount of ice remains

4. Spray with 70% EtOH and wipe off before returning to the hood

5. To each cryovial, add 1ml of R10 dropwise to each cryovial

6. Transfer the 2ml PBMC sample from the cryovials into the 50ml conicals

7.  Invert 3x to mix

8. Centrifuge at 350g for 10 min

9. Pour off the supernatant, do not shake to allow some volume to remain

10. Gently swirl the 50mL conical in remaining volume to loosen pellet

11. Add 10mL pre-warmed R10 and resuspend by pipetting 10 times. Mix sample carefully but
thoroughly to break up any cell clumps.

12. Centrifuge at 350g for 10 min

13. Pour off the supernatant, do not shake to allow some volume to remain

14. Gently swirl the 50mL conical in remaining volume to loosen pellet

15. Add 10mL pre-warmed R10 and resuspend by pipetting 10 times. Mix sample carefully but
thoroughly to break up any cell clumps

16.    COUNTING & VIABILITY:
a)      Perform a cell count using the Countess II to determine PBMC viability & recovery
b)      Add 10uL Trypan Blue to well of mixing plate
c)      Add 10uL Cells, pipette up and down
d)      Remove 10uL of cell mix and dispense into Countess slide
e)      Wait 30 sec
f)       Calculate total cells and viability
g)      Insert into Countess II to calculate total cells and viability
SECTION: Protocol
8.2. Overnight – Stim
Cells have been plated at 2x106 cells/well in 200uL R10 and rested for >6hours

2. For all wells (Stim and Unstim), add BFA/Monensin at 
a.      BFA: 0.23 uL per test
b.      Monensin: 0.16 uL per test
c.      CD107a antibody: 0.313uL per test

3. For Stim plates, add CEF PepMix at
a.     PepMix CEFX Ultra SuperStim Pool MHC-II Subset: 0.45uL per test
b.     PepMix CEF Pool (extended) : 0.45uL per test

4. Gently plate by vortexing at medium speed for 3 sec

5. Incubate cells at 37º C, 5% CO2 for 6 hours

6. At 6 hours (9am), wrap plate in Parafilm and place in 4C refrigerator and proceed with staining

 
  
  Stim Mixes
  
  
  # Tubes:
  
  
  16
  
  
  16
  
  
  
   
  
  
  uL per test
  
  
  Stim
  
  
  UnStim
  
  
  
  BFA
  
  
  0.23
  
  
  4.232
  
  
  4.232
  
  
  
  Monensin
  
  
  0.16
  
  
  2.944
  
  
  2.944
  
  
  
  CEFX ultra MHC II
  
  
  0.45
  
  
  8.28
  
  
  -
  
  
  
  CEF Pool Ext (MHC I)
  
  
  0.45
  
  
  8.28
  
  
  -
  
  
  
  CD107a
  
  
  0.313
  
  
  5.7592
  
  
  5.7592
  
  
  
  R10
  
  
  23.5
  
  
  432.4
  
  
  432.4
  
  
  
  Total
  
  
  25.103
  
  
   
  
  
   
  
  
  
  Add 25uL Mix to 200uL Cells for
  total 225uL
  
  
  
  
  


SECTION: Protocol
8.3. Day 2 – Stain

Keep everything as cold as much as possible
Keep everything covered as much as possible; work in dark or incandescent light


Surface staining:

1. Remove plates from refrigerator

2. Centrifuge 400g x 3 min

3. Flick off supernatant and vortex gently
     
4. Add 47.5uL FACS wash to each well

5. Add 2.5uL TruStain FCX blocking to each well

6. Incubate 4C for 15 min

7. Prepare Surface Stain Mix in PBS with 10uL BSB Plus, set aside

8. Add 50uL of Surface Stain Mix in PBS to each well and gently mix
     
9. Incubate in 4C fridge for 30 min, covered in foil
 
10. Add 100uL cold FACS wash & mix by pipetting up and down x3

11. Centrifuge 400g x 3 min

12. Flick off supernatant and vortex gently

13. Add 200uL cold FACS wash to each well
 
14. Centrifuge 400g x 3 min
 
15. Flick off supernatant and vortex gently

Intracellular cytokine staining (ICS): 

  

16. Add 100uL BD Cytofix/Cytoperm solution to each well.
a.     NOTE: mix well with cells

17. Incubate on ice for 20 min, covered in foil
a.     NOTE: do not over-incubate (Cytofix is toxic to cells)
    
18. Prepare ICS Stain Mix in 1X Perm Wash with 10uL BSB Plus, set aside
    
19. Add 100uL cold 1X Perm Wash solution

20. Centrifuge 400g x 3 min 
     
21. Flick off supernatant and vortex gently
    
22. Add 200uL cold 1X Perm Wash solution

23. Centrifuge 400g x 3 min
     
24. Flick off supernatant and vortex gently
    
25. Add 50uL of the ICS Mix in Perm Wash to each well and gently mix
 
26. Incubate at 4° in refrigerator x 30 min, covered in foil
   
27. Add 150uL cold 1X Perm Wash solution
 
28. Centrifuge 400g x 3 min

29. Flick off supernatant and vortex gently
  
30. Add 200uL cold 1X Perm Wash solution
  
31. Centrifuge 400g x 3 min

32. Flick off supernatant and vortex gently

33. Add 200uL cold 1X Perm Wash solution

34. Centrifuge 400g x 3 min

35. Flick off supernatant and vortex gently

36. Add 200uL 1% PFA

37. Transfer samples to bullet tubes, cover with aluminum foil, store at 4°C, & acquire within 6 hours

 
 
  
  Staining Step
  
  
  Specificity
  
  
  Fluor
  
  
  Vendor
  
  
  Cat#
  
  
  Clone
  
  
  Isotype
  
  
  Conc
  ug/mL
  
  
  MRA
  uL
  
  
  Titered
  uL
  
  
  
  Stim
  
  
  CD107a
  
  
  PE
  
  
  BL
  
  
  328608
  
  
  H4A3
  
  
  IgG1 k
  
  
  400
  
  
  5
  
  
  0.313
  
  
  
  Surface
  
  
  KLRG1
  
  
  BV421
  
  
  BL
  
  
  367706
  
  
  SA231A2
  
  
  IgG2a k
  
  
  100
  
  
  5
  
  
  1.25
  
  
  
  Surface
  
  
  CD45RA
  
  
  Pacific Blue
  
  
  BL
  
  
  304118
  
  
  H100
  
  
  IgG2b k
  
  
  500
  
  
  1
  
  
  0.5
  
  
  
  Surface
  
  
  CD8
  
  
  BV570
  
  
  BL
  
  
  301038
  
  
  RPA-T8
  
  
  IgG1 k
  
  
  100
  
  
  5
  
  
  2.5
  
  
  
  Surface
  
  
  CD127
  
  
  BV605
  
  
  BL
  
  
  351334
  
  
  A019D5
  
  
  IgG1 k
  
  
  100
  
  
  5
  
  
  2.5
  
  
  
  Surface
  
  
  CD56
  
  
  BV650
  
  
  BL
  
  
  362532
  
  
  5.1H11
  
  
  IgG1 k
  
  
  100
  
  
  5
  
  
  1.25
  
  
  
  Surface
  
  
  CCR7
  
  
  BV711
  
  
  BL
  
  
  353228
  
  
  G043H7
  
  
  IgG2a k
  
  
  100
  
  
  5
  
  
  5
  
  
  
  Surface
  
  
  CD27
  
  
  BV750
  
  
  BL
  
  
  302850
  
  
  O323
  
  
  IgG1 k
  
  
  100
  
  
  5
  
  
  2.5
  
  
  
  Surface
  
  
  PD1
  
  
  VioBright515
  
  
  Miltenyi
  
  
  130-120-386
  
  
  REA1165
  
  
  rHu
  IgG1
  
  
   
  
  
  2
  
  
  2
  
  
  
  Surface
  
  
  NKG2A
  
  
  PE-Vio615
  
  
  Miltenyi
  
  
  130-120-035
  
  
  REA110
  
  
  rHu IgG1
  
  
  
  2
  
  
  1
  
  
  
  Surface
  
  
  CD16
  
  
  PerCP-Cy5.5
  
  
  BL
  
  
  302028
  
  
  3G8
  
  
  IgG1 k
  
  
  200
  
  
  5
  
  
  2.5
  
  
  
  Surface
  
  
  CD38
  
  
  PerCp-eFluor710
  
  
  TF
  
  
  46-0388-42
  
  
  HB7
  
  
  IgG1 k
  
  
  120
  
  
  5
  
  
  5
  
  
  
  Surface
  
  
  CD19
  
  
  SparkNIR685
  
  
  BL
  
  
  302270
  
  
  HIB19
  
  
  IgG1 k
  
  
  100
  
  
  5
  
  
  1.25
  
  
  
  Surface
  
  
  CD14
  
  
  SparkNIR685
  
  
  BL
  
  
  367150
  
  
  63D3 
  
  
  IgG1 k
  
  
  200
  
  
  5
  
  
  0.156
  
  
  
  Surface
  
  
  Zombie nIR
  
  
  Zombie nIR
  
  
  BL
  
  
  423105
  
  
  -
  
  
  -
  
  
  -
  
  
  1
  
  
  0.4
  
  
  
  Surface
  
  
  HLA-DR
  
  
  APC-Fire810
  
  
  BL
  
  
  307674
  
  
  L243
  
  
  IgG2a k
  
  
  50
  
  
  5
  
  
  2.5
  
  
  
  ICS
  
  
  CD3
  
  
  BV480
  
  
  BD
  
  
  566105
  
  
  UCHT1
  
  
  IgG1 k
  
  
  200
  
  
  5
  
  
  2.5
  
  
  
  ICS
  
  
  CD4
  
  
  PerCP
  
  
  BD
  
  
  550631
  
  
  L200
  
  
  IgG1 k
  
  
  6.3
  
  
  20
  
  
  5
  
  
  
  ICS
  
  
  IFN-g
  
  
  PE-Cy7
  
  
  BL
  
  
  502528
  
  
  4S.B3
  
  
  IgG1 k
  
  
  50
  
  
  5
  
  
  0.313
  
  
  
  ICS
  
  
  IL-2
  
  
  APC
  
  
  BL
  
  
  500310
  
  
  MQ1-17H12
  
  
  Rat IgG2a k
  
  
  25
  
  
  5
  
  
  0.313
  
  
  
  ICS
  
  
  TNFa
  
  
  Alexa700
  
  
  BL
  
  
  502928
  
  
  MAb11
  
  
  IgG1 k
  
  
  100
  
  
  5
  
  
  0.63