Aug 17, 2023

Public workspaceU54 SCENT Intracellular Staining (ICS) Senescence Flow Cytometry Panel

DOI
dx.doi.org/10.17504/protocols.io.rm7vzxxk5gx1/v1
  • 1Duke University, Division of Pulmonary and Critical Care Medicine
Valerie Bekker
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DOI: dx.doi.org/10.17504/protocols.io.rm7vzxxk5gx1/v1
Protocol Citation: Zachary R Healy, David Murdoch, Alicia Cooper-Volkheimer 2023. U54 SCENT Intracellular Staining (ICS) Senescence Flow Cytometry Panel. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzxxk5gx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 17, 2023
Last Modified: August 17, 2023
Protocol Integer ID: 86602
Abstract
This protocol describes the Intracellular Staining (ICS) Senescence Flow Cytometry Panel

Materials
Reagents & Materials:
  • Pipettes and Tips for 1-1000uL
  • 96 Well Round Bottom Plates: Costar Cat #3799
  • Bullet Tubes: Costar Cat #4401
  • BSB Plus:BD Cat #566385
  • TruStain FcX: BioLegend Cat #422302
  • Monensin: BD Cat # 51-2092KZ, Store undiluted at 4°C.
  • GolgiPlug - Brefeldin A Solution (“BFA”): BD Cat #555029, Store @ 4°C
  • dPBS: Invitrogen Cat #: 41190-250
FBS Aliquot Prep
FBS Aliquot Prep
FBS (hiFBS): Gemini Bio Products Cat #100-106 1L
Prepare FBS Aliquots:
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.
Aliquot the 500mL FBS Bottle in 50mL aliquots (a total of ten 50mL hi-FBS Aliquots)
Label the aliquots with Batch#, Expiration Date, Aliquot Date
Store 50mL aliquots @ -20ºC until expiration date or for up to 2 freeze/thaw cycles
FACS Wash
FACS Wash
FACS Wash w/ EDTA (D-PBS with 0.5% FBS + 2 mM EDTA): Invitrogen Cat # 41190-250
Remove 4.5mL PBS from a 500mL bottle
Add 2.5mL of thawed FBS
Add 2mL of 0.5M EDTA solution (Catalog #: E7889-100ML)
Label the bottle with preparer's initials and expiration date (one month from preparation)
Store @ 4°C
Pen-Strep-Glut (PSG)
Pen-Strep-Glut (PSG)
Pen-Strep-Glut (PSG) (L-Glutamine-Penicillin-Streptomycin Soln): Sigma Cat#: G6784-100ML
Thaw 100mL bottle
Aliquot into 10mL into 15mL conicals (total of ten 15mL conicals)
Label the aliquots with Batch#, Expiration Date, Aliquot Date
Store @ -20°C
R10FBS Media Preparation
R10FBS Media Preparation
R10FBS Media Preparation (“R10”)
Remove 55mL RPMI from a 500mL bottle of RPMI
Add 50mL aliquot of thawed, hiFBS
Add 5mL aliquot of thawed Pen-Strep-Glut
Label the bottle with preparer's initials and expiration date (one month from preparation)
Cytofix/CytopermSolution (10X) with GolgiStop (monensin)
Cytofix/CytopermSolution (10X) with GolgiStop (monensin)
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
Formaldehyde Solution
Formaldehyde Solution
1% Formaldehyde Solution (“1% Fix”) (“PFA”)
Reagents:
  • 10% Formalin
  • PBS
Add 5 mL 10% Formalin to a sterile 50 mL centrifuge conical tube
Add 45 mL PBS
Label the bottle with reagent name, initials & expiration date (one month from preparation)
Store 1% Fix at Room Temperature (18-25°C) for up 1 month
PepMixes
PepMixes
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
Resuspend peptide pellet [25ug] in 50uL of DMSO [500ug/mL]                
  • Add 10uL DMSO at a time, with vortexing to resuspend
Aliquot and store at -20°C until day of use
On day of use, use 0.45uL per test
Protocol
Protocol
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
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
Overnight – Stim
  1. 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

ABCD
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
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

ABCDEFGHIJ
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