Processing of pediatric whole blood samples for single cell analysis

Label an EDTA blood collection tube with study/patient ID.

After obtaining informed consent from family and/or patient, collect blood sample into EDTA tube.

Whole blood samples must remain at Room temperature and be processed in the laboratory within 0h 30m 0s to 1h 0m 0s of the procedure.

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.

Centrifuge the remaining whole blood sample at 700x g to separate out plasma.

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).

Fill a 15mL tube with 2mL of Ficoll plaque plus and layer the diluted blood onto the surface of the Ficoll solution.

Centrifuge the layered cell suspension at 400x g .

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 400x g,4°C .

There is an option here to harvest granulocytes for downstream analysis, including genomics, which we have detailed below.

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.

Discard supernatant and resuspend the cell pellet in 3mL RPMI 2% FCS.

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.

Load 10µL of stained cells onto a TC-20 slide (Bio-Rad) chamber and count. Record viability, total cell count, and live cell count.

Top up the cell suspension to 10 mL with RPMI 2% FCS and centrifuge at 400x g .

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.

Immediately place cryogenic vials into an isopropanol freezing container (e.g. Nalgene^® Mr. Frosty) or Cool Cell (Corning) and transfer to -80°C overnight.

For long-term storage, transfer the vials to liquid nitrogen.

Resuspend whole blood in 2mL red cell lysis buffer for 0h 10m 0s. Centrifuge at 500x g,4°C and discard supernatant.

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

400x g,4°C

Resuspend cells in 25µL FACS buffer and add 15µL FC-block for 0h 5m 0s at Room temperature.

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.

tonsil_adenoid_blood_panel.pdf

Add 10µL of Brilliant Stain Buffer (Becton Dickinson) and then add 25µL of Cocktail 1A made up at 3X concentration and incubate for 0h 10m 0s

Then, directly add cockta made up at 2X concentration 1:1 with cells and incubate for 0h 30m 0s

Following staining, wash cells with 2mL FACS buffer and centrifuge at 400x g,4°C and resuspend cells in 100µL FACS buffer for acquisition on a flow cytometer (here, a Cytek 5L aurora).