TITLE: Whole blood assay-Scrub typhus for flow cytometer
AUTHORS: Manutsanun Inthawong

[DESCRIPTION]
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 sterility
PBS
·Made with PBS tablets from SIGMA, and dH2O.1 tablet in 100ml water.



[STEPS]
SECTION: Day 1. Whole blood stimulation for scrub typhus study
1. 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
 

 

2. 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)
3. Add 400  µL  of lithium heparinised blood to each tube, cap  tightly, vortex.

 

SECTION: Day 2. Harvest stimulated white blood cells and cryopreservation
5. Add 25 µL Brefeldin A (10 µg/mL)  

SECTION: Day 2. Harvest stimulated white blood cells and cryopreservation
7. Stain with Live-Dead Staining dye (Near IR) 1 µl which enables discrimination of live from dead cells in subsequent flow cytometry analysis  

SECTION: Day 2. Harvest stimulated white blood cells and cryopreservation
8. Incubate on ice  for 20 min.
SECTION: Day 2. Harvest stimulated white blood cells and cryopreservation
9. Wash the sample once with Phosphate buffered saline (PBS) and centrifuge at 500 g for 5 min.

 

SECTION: Day 2. Harvest stimulated white blood cells and cryopreservation
11. Red blood cell lysis (RBC) was performed by adding 3 ml of 1x FACS lysing solution  

SECTION: Day 2. Harvest stimulated white blood cells and cryopreservation
10. Remove  supernatant by carefully pipetting without disturbing the pellet (RBC) 
SECTION: Day 2. Harvest stimulated white blood cells and cryopreservation
12. Incubate at room  temperature for 10 min. (to help RBC lysis), vortex for 30 s.
SECTION: Day 2. Harvest stimulated white blood cells and cryopreservation
13. Spin in centrifuge for 5 min at 500 rcf
SECTION: Day 2. Harvest stimulated white blood cells and cryopreservation
14. Remove supernatant by pouring off then vortex to resuspend pellet for 10 s
SECTION: Day 2. Harvest stimulated white blood cells and cryopreservation
15. Adding 3 ml of 1x FACS lysing solution, vortex for 30 s. 
SECTION: Day 2. Harvest stimulated white blood cells and cryopreservation
16. Spin in centrifuge for 5 min at 500 rcf
SECTION: Day 2. Harvest stimulated white blood cells and cryopreservation
17. Remove supernatant by pouring off then vortex to resuspend pellet for 10 s
SECTION: Day 2. Harvest stimulated white blood cells and cryopreservation
18. 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
 

SECTION: Intracellular cytokine staining (ICS) for Flow cytometry
19. To do ICS, cryopreserved stimulated whole blood samples were slowly thawed in a 37°C water bath
4. Incubate at 37 °C  for 18-20 hours in CO2 incubator or in a water bath  (optional).
SECTION: Day 2. Harvest stimulated white blood cells and cryopreservation
6. Incubate at 37 °C  for further 4 hours in CO2 incubator or in a water bath  (optional).
SECTION: Intracellular cytokine staining (ICS) for Flow cytometry
20. The cells are then undergo fixation and permeabilization using Cytofix (BD Biosciences,  ) and Cytoperm/Wash (BD Biosciences  ) according to the manufacturer’s instructions 

SECTION: Intracellular cytokine staining (ICS) for Flow cytometry
21. 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).
SECTION: Intracellular cytokine staining (ICS) for Flow cytometry
22. Acquisition of a minimum of 100,000 cells per sample was performed on a MACSQuant 10 analyzer (Miltenyi Biotec). 
SECTION: Intracellular cytokine staining (ICS) for Flow cytometry
21.1. After adding antibody for surface staining, the cells are incubated for 30 min at 4 °C 

SECTION: Intracellular cytokine staining (ICS) for Flow cytometry
21.2. Washing the cells in centrifuge for 5 min at 500 rcf
SECTION: Intracellular cytokine staining (ICS) for Flow cytometry
21.3. Remove supernatant by pouring off then vortex to resuspend pellet for 10 s
SECTION: Intracellular cytokine staining (ICS) for Flow cytometry
21.4. After adding antibody for intracellular staining, the cells are incubated for 30 min at 4 °C 
SECTION: Intracellular cytokine staining (ICS) for Flow cytometry
21.5. Washing the cells in centrifuge for 5 min at 500 rcf
SECTION: Intracellular cytokine staining (ICS) for Flow cytometry
21.6. Remove supernatant by pouring off then vortex to resuspend pellet for 10 s
SECTION: Intracellular cytokine staining (ICS) for Flow cytometry
21.7. Resuspend cells with 200 µl fixation buffer (BD Bioscience, Cat 554655) for subsequent flow cytometric analysis.