TITLE: Processing of pediatric whole blood samples for single cell analysis
AUTHORS: Liam Gubbels, Shivanthan Shanthikumar, Melanie R Neeland

[DESCRIPTION]
This protocol describes the collection, processing, and cryopreservation of whole blood samples for downstream single-cell analysis.

[GUIDELINES]
This is an experimental protocol for the processing of whole blood samples collected from children. Sample collection must have and be compliant with Human Ethics Committee approval.

[STEPS]
SECTION: COLLECTION OF WHOLE BLOOD
1. Label an EDTA blood collection tube with study/patient ID. 
SECTION: COLLECTION OF WHOLE BLOOD
3. Whole blood samples must remain at Room temperature and be processed in the laboratory within 30 min to 60 min of the procedure.

SECTION: COLLECTION OF WHOLE BLOOD
2. After obtaining informed consent from family and/or patient, collect blood sample into EDTA tube.

SECTION: PROCESSING OF WHOLE BLOOD TO PBMC CELL SUSPENSION
4. First, take 100µL whole blood and place into a pre-labelled tube for downstream single cell analysis, such as flow cytometry detailed herein from step 16.
SECTION: PROCESSING OF WHOLE BLOOD TO PBMC CELL SUSPENSION
11. Discard supernatant and resuspend the cell pellet in 3mL RPMI 2% FCS.  
SECTION: PROCESSING OF WHOLE BLOOD TO PBMC CELL SUSPENSION
10.1. While the mononuclear cells are spinning, harvest the granulocyte layer at the interphase between the erythrocytes and the Ficoll solution and place into a labelled cryovial. Add 1mL of RNAlater solution to the collected granulocytes and pipette mix thoroughly. Immediately transfer the granulocytes to -80 °C for long-term storage. 
SECTION: PROCESSING OF WHOLE BLOOD TO PBMC CELL SUSPENSION
10. There is an option here to harvest granulocytes for downstream analysis, including genomics, which we have detailed below. 
SECTION: PROCESSING OF WHOLE BLOOD TO PBMC CELL SUSPENSION
9. Once the spin is complete, carefully aspirate the mononuclear layer at the interface between the RPMI 2% FCS and the Ficoll solution into a new 15mL tube. Top up the cell suspension to 10mL using RPMI 2% FCS and centrifuge 400 x g, 5 min, 4 °C .

 


SECTION: PROCESSING OF WHOLE BLOOD TO PBMC CELL SUSPENSION
8. Centrifuge the layered cell suspension at 400 x g, 30 min .
SECTION: PROCESSING OF WHOLE BLOOD TO PBMC CELL SUSPENSION
7. Fill a 15mL tube with 2mL of Ficoll plaque plus and layer the diluted blood onto the surface of the Ficoll solution. 

 

SECTION: PROCESSING OF WHOLE BLOOD TO PBMC CELL SUSPENSION
6. Aspirate plasma layer and store in 2mL cryotubes, then transfer to -80 °C for long-term storage. Dilute the plasma-depleted blood to its initial volume using room temperature RPMI supplemented with 2% heat-inactivated fetal calf serum (referred to as RPMI 2% FCS). 
SECTION: PROCESSING OF WHOLE BLOOD TO PBMC CELL SUSPENSION
5. Centrifuge the remaining whole blood sample at 700 x g, 7 min to separate out plasma. 
SECTION: PROCESSING OF WHOLE BLOOD TO PBMC CELL SUSPENSION
12. Prepare cell suspension for cell counting. Here, we use trypan blue and a Bio-Rad TC-20 cell counter. Take 10µL of the cell suspension and place into a microcentrifuge tube for cell counting. Add 10µL of trypan blue to the microcentrifuge tube and mix thoroughly. 
SECTION: PROCESSING OF WHOLE BLOOD TO PBMC CELL SUSPENSION
12.1. Load 10µL of stained cells onto a TC-20 slide (Bio-Rad) chamber and count. Record viability, total cell count, and live cell count. 

 

SECTION: CRYOPRESERVATION OF PBMC
14. Discard supernatant and resuspend cells at a ratio of 1:1 in RPMI 2% FCS and freeze solution (heat-inactivated FCS + 15% DMSO) such that cells are frozen between 1-10 million cells/mL. Transfer cells to cryogenic vial.

 

SECTION: CRYOPRESERVATION OF PBMC
16. For long-term storage, transfer the vials to liquid nitrogen.
SECTION: CRYOPRESERVATION OF PBMC
15. Immediately place cryogenic vials into an isopropanol freezing container (e.g. Nalgene‱ Mr. Frosty) or Cool Cell (Corning) and transfer to -80 °C overnight.
SECTION: PREPARATION OF WHOLE BLOOD CELLS FOR FLOW CYTOMETRY
17. Resuspend whole blood in 2mL red cell lysis buffer for 10 min. Centrifuge at 500 x g, 5 min, 4 °C and discard supernatant.  
SECTION: PREPARATION OF WHOLE BLOOD CELLS FOR FLOW CYTOMETRY
18. Resuspend cell suspension for fixable viability staining according to manufacturers' instructions (e.g. the Fixable UV Blue Stain from Invitrogen/ThermoFisher from Invitrogen/ThermoFisher). 

Following the required incubation, stop the reaction by the addition of 1mL staining buffer (2% heat-inactivated FCS in PBMS 2 mM EDTA, herein referred to as FACS buffer) and centrifuge at 
400 x g, 5 min, 4 °C 
SECTION: PREPARATION OF WHOLE BLOOD CELLS FOR FLOW CYTOMETRY
19. Resuspend cells in 25µL FACS buffer and add 15µL FC-block for 5 min at Room temperature.
SECTION: PREPARATION OF WHOLE BLOOD CELLS FOR FLOW CYTOMETRY
20. The next steps will depend on the requirements for your specific panel. As an example, we have attached our 31-plex spectral cytometry panel that we routinely use on whole blood cells. All of the following steps are related to this panel.

  
SECTION: PREPARATION OF WHOLE BLOOD CELLS FOR FLOW CYTOMETRY
22. Then, directly add cockta made up at 2X concentration 1:1 with cells and incubate for 30 min 
SECTION: PREPARATION OF WHOLE BLOOD CELLS FOR FLOW CYTOMETRY
23. Following staining, wash cells with 2mL FACS buffer and centrifuge at 400 x g, 5 min, 4 °C  and resuspend cells in 100µL FACS buffer for acquisition on a flow cytometer (here, a Cytek 5L aurora). 

 

SECTION: PROCESSING OF WHOLE BLOOD TO PBMC CELL SUSPENSION
13. Top up the cell suspension to 10 mL with RPMI 2% FCS and centrifuge at 400 x g, 5 min .
SECTION: PREPARATION OF WHOLE BLOOD CELLS FOR FLOW CYTOMETRY
21. Add 10µL of Brilliant Stain Buffer (Becton Dickinson) and then add 25µL of Cocktail 1A made up at 3X concentration and incubate for 10 min