May 07, 2024
Processing of pediatric whole blood samples for single cell analysis
- Liam Gubbels1,
- Shivanthan Shanthikumar1,
- Melanie R Neeland1
- 1Murdoch Children's Research Institute
- earlyAIR
Protocol Citation: Liam Gubbels, Shivanthan Shanthikumar, Melanie R Neeland 2024. Processing of pediatric whole blood samples for single cell analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov19y37lr2/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: April 01, 2024
Last Modified: May 07, 2024
Protocol Integer ID: 97621
Keywords: single-cell, flow cytometry, respiratory, Paediatric, processing of pediatric whole blood sample, pediatric whole blood sample, samples for downstream single cell analysis, single cell analysis, downstream single cell analysis, cryopreservation of whole blood sample, single cell analysis this protocol, whole blood sample, cell analysis, cryopreservation, cell, blood
Funders Acknowledgements:
Chan Zuckerberg Initiative
Grant ID: 2021-237883
Abstract
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.
Materials
RPMI-1640Merck MilliporeSigma (Sigma-Aldrich)Catalog #R5886
Sample tube, EDTA K3E, 10 ml, cap redSarstedtCatalog #26.358
Fetal Bovine Serum
1X PBS (Phosphate-buffered saline )
DMSO (dimethyl sulfoxide)Merck MilliporeSigma (Sigma-Aldrich)Catalog #D8418
conical tubes, 50ml
conical tubes, 15ml
1.5 mL Eppendorf tubes
Screw cap micro tube, 2 mLSarstedtCatalog #72.694.600
Ficoll Paque PLUSGE HealthcareCatalog #17144003-500 ml
0.4% Trypan blue solution
Cell Counting Slides for TC10™/TC20™ Cell Counter Dual-Chamber 5 x 30 slides 300 counts #1450015Bio-Rad LaboratoriesCatalog #1450015
FACS Tubes
Brilliant stain bufferBecton Dickinson (BD)Catalog #563794
Human TruStain FcX™ (Fc Receptor Blocking Solution)BioLegendCatalog #422301, 422302
cryovial
RNAlaterMerck MilliporeSigma (Sigma-Aldrich)Catalog #R0901-100ML
Flow antibodies: 
Flow cytometry panels.pdf301KB
Flow cytometry panels.pdf301KB Equipment
Aurora - 5L Configuration
NAME
Spectral Flow Cytometer
TYPE
Cytek Aurora
BRAND
N/A
SKU
5L Configuration
SPECIFICATIONS
Equipment
TC20 Automated Cell Counter
NAME
automated cell counter
TYPE
BioRad
BRAND
1450102
SKU
LINK
Troubleshooting
Safety warnings
Human samples should be processed in a laboratory with appropriate biosafety infrastructure and procedures
COLLECTION OF WHOLE BLOOD
1h 30m
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 00:30:00 to 01:00:00 of the procedure.
1h 30m
PROCESSING OF WHOLE BLOOD TO PBMC CELL SUSPENSION
47m
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 700 x g, Room temperature, 00:07:00 to separate out plasma.
7m
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.
Note
Layer the cell suspension slowly to prevent the Ficoll solution from mixing with the cells.
Centrifuge the layered cell suspension at 400 x g, Room temperature, 00:30:00 , 4 Acceleration and NO brake .
45m
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, 4°C, 00:05:00 .
Note
When collecting the cells, try to avoid Ficoll solution as much as possible.
5m
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.
Note
Cell counting can also be performed manually using a haemocytometer, or using other automated cell counters.
Top up the cell suspension to 10 mL with RPMI 2% FCS and centrifuge at 400 x g, Room temperature, 00:05:00 .
5m
CRYOPRESERVATION OF PBMC
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.
Note
If performing downstream single cell analysis on PBMC, prior to adding freeze solution is where you can allocate out the required number of fresh PBMCs.
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.
PREPARATION OF WHOLE BLOOD CELLS FOR FLOW CYTOMETRY
1h 10m
Resuspend whole blood in 2mL red cell lysis buffer for 00:10:00 at Room Temperature . Centrifuge at 500 x g, 4°C, 00:05:00 and discard supernatant.
15m
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, 4°C, 00:05:00
5m
Resuspend cells in 25µL FACS buffer and add 15µL FC-block for 00:05:00 at Room temperature .
5m
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.pdf127KB Add 10µL of Brilliant Stain Buffer (Becton Dickinson) and then add 25µL of Cocktail 1A made up at 3X concentration and incubate for 00:10:00 In the dark at room temperature
10m
Then, directly add cockta made up at 2X concentration 1:1 with cells and incubate for 00:30:00 In the dark at room temperature
30m
Following staining, wash cells with 2mL FACS buffer and centrifuge at 400 x g, 4°C, 00:05:00 and resuspend cells in 100µL FACS buffer for acquisition on a flow cytometer (here, a Cytek 5L aurora).
Note
Panels 1A and 1B were adapted from: "OMIP-069: Forty-Color Full Spectrum Flow Cytometry Panel for Deep Immunophenotyping of Major Cell Subsets in Human Peripheral Blood" and we thank the authors for their detailed methods.
Citation
LINK
5m