Aug 17, 2023
U54 SCENT T/NK Immunosenescence Profiling Flow Cytometry Panel
- Zachary R Healy1,
- David Murdoch1,
- Alicia Cooper-Volkheimer1
- 1Duke University, Division of Pulmonary and Critical Care Medicine
Protocol Citation: Zachary R Healy, David Murdoch, Alicia Cooper-Volkheimer 2023. U54 SCENT T/NK Immunosenescence Profiling Flow Cytometry Panel. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7ppm9gwz/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: 86607
Abstract
This protocol describes the T/NK Immunosenescence Profiling 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
- 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)
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 RT (18-25°C) for up 1 month
Protocol
Protocol
Overview
Thaw cells and distribute to plates. Stain immediately, then acquire flow data.
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
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
| A | B | C | D | E | F | G | H | I | |
| 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 |