Whole blood assay-Scrub typhus for flow cytometer
Manutsanun Inthawong
Abstract
This is an optimized new whole blood assay for studying scrub typhus-specific cellular responses specifically suited for work in remote areas. This assay is based on stimulation with a whole cell O. tsutsugamushi antigen (HI-WCA-OT) in order to study broad antigen-specific cellular immune responses.
We provide the preparation method (Heat inactivation) and the adequate concentration for the antigen (109 of O. tsutsugamushi DNA copies/ml) to obtain optimal measureable specific cellular immune responses in whole blood samples for use in a Biosafety level (BSL) 2 laboratory.
Before starring
Solutions to be made up
R0 ·490 ml RPMI 1640 (without glutamine)
·5 ml 10000 U/ml penicillin/streptomycin from aliquot.
·5 ml 200 mM L-Glutamine from aliquot.
·Label “R0” and write date, initials, and write PS and LG lot numbers on the bottle. Store at 4°C and replace after 1 month or if any concern about sterili PBS PBS
·Made with PBS tablets from SIGMA, and dH2O.1 tablet in 100ml water.
Steps
Day 1. Whole blood stimulation for scrub typhus study
Prepare pre-labelled 5 ml tubes to each contain 5 µl of “ COCKTAIL ” (co-stimulant) and NO Brefeldin A. Add the stimulants as follows to respective tubes as below
| A | B | C |
|---|---|---|
| HI-WCA-OT | 100 | prepared from 10E9 copies/ml of Live O. tsutsugamushi |
| SEB (positive control) | 100 | Dilute 1: 20 of stock SEB (1 mg/ml) in R10 Add 100 µl of this “SEB WBA” to each tube for final concentration of 10 µg/ml in 400 µl blood |
| R0 (negative control | 100 |
| A | B | C | D |
|---|---|---|---|
| CD28 | BD Bioscience | 340975 | 1 µg/ml |
| CD49d | BD Bioscience | 340976 | 1 µg/ml |
COCKTAIL (co-stimulant)Each provided as 200 µg in 0.01% azide in PBS; 1 mg/mL. Store at 4°C in the dark.Dilute 1: 10 of each in a 1 ml tube, combine 3 µl anti-CD283 µl anti-CD49d24 µl PBS. Label “COCKTAIL”For 500 µL blood; add 5 µl of the above “COCKTAIL” solution for a final concentration of 1 µg/mL each.
Each subject in the study needs 3 x 5 ml tubes labelled with their ID number and stimulant (antigen or control) i.e. “HI-WCA-OT”(Antigen of interest, Heat inactivated whole cell antigen of Orientia tsutsugamushi ) “SEB” (Positive control) or “R0” (Negative control)
Add 400 µL of lithium heparinised blood to each tube, cap tightly, vortex.
Incubate at 37°C for 18-20 hours in CO2 incubator or in a water bath (optional).
Day 2. Harvest stimulated white blood cells and cryopreservation
Add 25 µL Brefeldin A (10 µg/mL)
Incubate at 37°C for further 4 hours in CO2 incubator or in a water bath (optional).
Stain with Live-Dead Staining dye (Near IR) 1 µl which enables discrimination of live from dead cells in subsequent flow cytometry analysis
Incubate on ice for 20 min.
Wash the sample once with Phosphate buffered saline (PBS) and centrifuge at 500 g for 5 min.
Remove supernatant by carefully pipetting without disturbing the pellet (RBC)
Red blood cell lysis (RBC) was performed by adding 3 ml of 1x FACS lysing solution
Incubate at room temperature for 10 min. (to help RBC lysis), vortex for 30 s.
Spin in centrifuge for 5 min at 500 rcf
Remove supernatant by pouring off then vortex to resuspend pellet for 10 s
Adding 3 ml of 1x FACS lysing solution, vortex for 30 s.
Spin in centrifuge for 5 min at 500 rcf
Remove supernatant by pouring off then vortex to resuspend pellet for 10 s
Add 1 ml freezing mix to resuspended pellet and transfer 500 µl each into 2 labelled cryotubes (so that there is two tubes for each condition for freezing) followed by stepwise freezing to -80°C
Intracellular cytokine staining (ICS) for Flow cytometry
To do ICS, cryopreserved stimulated whole blood samples were slowly thawed in a 37°C water bath
The cells are then undergo fixation and permeabilization using Cytofix (BD Biosciences,
Cells were stained with the following fluorochrome conjugated antibodies to assess polyfunctionality and memory:
Surface staining: CD8 V450 (clone RPA-T8, Cat 560347), CD3 V500 (clone SP34-2, Cat 560770), CD4 FITC (clone RPA-T4, Cat 555346 and clone M-T477, Cat 556615), CD45RA-PE (clone HI100, Cat 555489) all from BD Biosciences.
Intracellular staining: IFN-gamma APC (clone B27, BD Biosciences, Cat 554702), IL-2 PE (clone N7.48A, Beckman, Cat IM2718U) and TNF PCP-Cy5.5 (clone MAb11, eBioscience, Cat 45-7349-42).
After adding antibody for surface staining, the cells are incubated for 30 min at 4°C
Washing the cells in centrifuge for 5 min at 500 rcf
Remove supernatant by pouring off then vortex to resuspend pellet for 10 s
After adding antibody for intracellular staining, the cells are incubated for 30 min at 4°C
Washing the cells in centrifuge for 5 min at 500 rcf
Remove supernatant by pouring off then vortex to resuspend pellet for 10 s
Resuspend cells with 200 µl fixation buffer (BD Bioscience, Cat 554655) for subsequent flow cytometric analysis.
Acquisition of a minimum of 100,000 cells per sample was performed on a MACSQuant 10 analyzer (Miltenyi Biotec).
