TITLE: U54 SCENT T/NK Immunosenescence Profiling Flow Cytometry Panel
AUTHORS: Zachary R Healy, David Murdoch, Alicia Cooper-Volkheimer

[DESCRIPTION]
This protocol describes the T/NK Immunosenescence Profiling 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: Formaldehyde Solution
5. 1% Formaldehyde Solution (“1% Fix”) (“PFA”)

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



SECTION: Protocol
6. Overview
Thaw cells and distribute to plates. Stain immediately, then acquire flow data.
SECTION: Protocol
6.1. 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)       Insert into Countess II to calculate total cells and viability

17. Centrifuge 350g x10 min
   
18. Decant supernatant and resuspend at 10x106 viable cells/mL R10
  
19. Aliquot 100uL of cells per well to plate (1x106 cells/well) for immediate staining
SECTION: Protocol
6.2. Staining

Keep everything as cold as much as possible
Keep everything covered as much as possible; work in dark or incandescent light
      
1. Centrifuge (400 x 3 min)

2. Flick off supernatant and vortex gently
      
3. Add 47.5uL FACS wash to each well
       
4. Add 2.5uL TruStain FCX blocking to each well
     
5. Incubate at 4C for 15 min
       
6. Prepare experiment-specific surface stain antibody mix in PBS with 10uL BSB Plus, set aside
       
7. Add 100uL of antibody mix to each well and gently vortex
       
8. Incubate in 4C fridge for 30 min

9. Add 100uL FACS wash to each well
     
10. Centrifuge (400g x 3 min)
   
11. Flick off supernatant and vortex gently

12. Add 200uL FACS wash to each well

13. Centrifuge (400g x 3 min)
   
14. Flick off supernatant and vortex gently

15. Add 200uL FACS wash to each well
 
16. Centrifuge (400g x 3 min)
 
17. Flick off supernatant and vortex gently

18. Add 200uL 1% PFA
  
19. Transfer samples to bullet tubes, cover with aluminum foil, store at 4°C, & acquire within 6 hours


 
  
  Specificity
  
  
  Fluor
  
  
  Vendor
  
  
  Cat#
  
  
  Clone
  
  
  Isotype
  
  
  Conc
  ug/mL
  
  
  MRA
  uL
  
  
  Titered
  uL
  
  
  
  KLRG1
  
  
  BV421
  
  
  BioLegend
  
  
  367706
  
  
  SA231A2
  
  
  IgG2a
  k
  
  
  100
  
  
  5
  
  
  1.25
  
  
  
  CD45RA
  
  
  Pacific
  Blue
  
  
  BioLegend
  
  
  304118
  
  
  H100
  
  
  IgG2b
  k
  
  
  500
  
  
  1
  
  
  0.5
  
  
  
  CD4
  
  
  BV480
  
  
  BD
  
  
  566104
  
  
  SK3
  
  
  IgG1
  k
  
  
  50
  
  
  5
  
  
  1.25
  
  
  
  CD8
  
  
  BV570
  
  
  BioLegend
  
  
  301038
  
  
  RPA-T8
  
  
  IgG1
  k
  
  
  100
  
  
  5
  
  
  2.5
  
  
  
  CD45RO
  
  
  BV605
  
  
  BioLegend
  
  
  304238
  
  
  UCHL1
  
  
  IgG2a
  k
  
  
  100
  
  
  5
  
  
  5
  
  
  
  CD56
  
  
  BV650
  
  
  BioLegend
  
  
  362532
  
  
  5.1H11
  
  
  IgG1
  k
  
  
  100
  
  
  5
  
  
  1.25
  
  
  
  CX3CR1
  
  
  BV711
  
  
  BioLegend
  
  
  341630
  
  
  2A9-1
  
  
  Rat
  IgG2b k
  
  
  100
  
  
  5
  
  
  2.5
  
  
  
  CCR7
  
  
  BV785
  
  
  BioLegend
  
  
  353230
  
  
  G043H7
  
  
  IgG2a
  k
  
  
  200
  
  
  5
  
  
  5
  
  
  
  PD1
  
  
  
  VioBright515
  
  
  
  Miltenyi
  
  
  
  130-120-386
  
  
  
  REA1165
  
  
  
  rHu IgG1
  
  
  
  
  
  2
  
  
  
  2
  
  
  
  
  CD3
  
  
  AF532
  
  
  Thermo
  Fisher
  
  
  58-0038-42
  
  
  UCHT1
  
  
  IgG1
  k
  
  
  50
  
  
  5
  
  
  5
  
  
  
  CD127
  
  
  PE
  
  
  BioLegend
  
  
  351304
  
  
  A019D5
  
  
  IgG1
  k
  
  
  500
  
  
  5
  
  
  2.5
  
  
  
  NKG2A
  
  
  PE-Vio615
  
  
  Miltenyi
  
  
  130-120-035
  
  
  REA110
  
  
  rHu
  IgG1
  
  
  
  2
  
  
  1
  
  
  
  CD27
  
  
  PE-Cy5
  
  
  BioLegend
  
  
  356438
  
  
  M-T271
  
  
  IgG1
  k
  
  
  50
  
  
  5
  
  
  1.25
  
  
  
  CD16
  
  
  PerCP-Cy5.5
  
  
  BioLegend
  
  
  302028
  
  
  3G8
  
  
  IgG1
  k
  
  
  200
  
  
  5
  
  
  2.5
  
  
  
  CD38
  
  
  PerCP-eFluor710
  
  
  TF
  
  
  46-0388-42
  
  
  HB7
  
  
  IgG1
  k
  
  
  120
  
  
  5
  
  
  5
  
  
  
  CD25
  
  
  PE-Cy7
  
  
  BioLegend
  
  
  356108
  
  
  M-A251
  
  
  IgG1
  k
  
  
  100
  
  
  5
  
  
  5
  
  
  
  CD14
  
  
  APC
  
  
  BioLegend
  
  
  367118
  
  
  63D3
  
  
  IgG1
  k
  
  
  200
  
  
  5
  
  
  2.5
  
  
  
  CD19
  
  
  APC
  
  
  BioLegend
  
  
  363006
  
  
  SJ25C1
  
  
  IgG1
  k
  
  
  50
  
  
  5
  
  
  2.5
  
  
  
  CD28
  
  
  AF647
  
  
  BioLegend
  
  
  302954
  
  
  CD28.2
  
  
  IgG1
  k
  
  
  100
  
  
  5
  
  
  5
  
  
  
  Zombie
  nIR
  
  
  Zombie
  nIR
  
  
  BioLegend
  
  
  423105
  
  
  -
  
  
  -
  
  
  -
  
  
  1
  
  
  0.4
  
  
  
  CD95
  
  
  APC-Fire810
  
  
  BioLegend
  
  
  305663
  
  
  DX2
  
  
  IgG1
  k
  
  
  200
  
  
  5
  
  
  1.25