Mar 20, 2025
Enrichment of Reticulocytes from Whole Blood
- Awodabon Fomukong Hanneda1,2,3,
- Abubakar Sani1,2,4,
- Idowu Aimola1,2,
- Mamman Mohammed1,5,
- Halima Bello Manga6,
- Gloria Chechet1,2,
- uyomoses1,2,
- samsonbabareuben2,
- Abduljabar Musa2,
- Okoro Peculiar Nwanyibunwa1,2
- 1Africa Center of Excellence for Neglected Tropical Diseases and Forensic Biotechnology, Ahmadu Bello University, Zaria, Nigeria.;
- 2Department of Biochemistry, Ahmadu Bello University, Zaria, Nigeria.;
- 3Department of Biochemistry, Capital City University Kano, Kano State, Nigeria.;
- 4Department of Science Laboratory Technology, Federal Polytechnic Daura, Katsina State, Nigeria.;
- 5Department of Veterinary Pharmacology & Toxicology, Ahmadu Bello University, Zaria, Nigeria.;
- 6Barau Dikko Teaching Hospital, Kaduna, Nigeria.
Protocol Citation: Awodabon Fomukong Hanneda, Abubakar Sani, Idowu Aimola, Mamman Mohammed, Halima Bello Manga, Gloria Chechet, uyomoses, samsonbabareuben, Abduljabar Musa, Okoro Peculiar Nwanyibunwa 2025. Enrichment of Reticulocytes from Whole Blood . protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l29xn3v1y/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: March 10, 2025
Last Modified: March 20, 2025
Protocol Integer ID: 124091
Keywords: Reticulocytes, Single-cells, Red blood cells
Funders Acknowledgements:
African Academy of Science
Grant ID: APTI-18-02
Abstract
Reticulocytes are a group of immature RBCs produced in the bone marrow and released into the bloodstream where they mature into RBCs within one to two days. They play a crucial role in assessing bone health and many haematological diseases. Isolating viable reticulocytes for use in single-cell transcriptomics has been a challenge due to their similarity to mature RBCs. Their low concentration and continuous maturation to RBCs with time also poses a great challenge. In this study, we designed a protocol for enrichment of reticulocytes from whole blood, with a high yield and viability for single-cell transcriptomic studies.
Attachments
Guidelines
This protocol is used to enrich and obtain high concentrations of viable reticulocytes, which could be useful for single cell transcriptomics studies.
Materials
- EDTA tubes (BioRapid)
- Falcon tubes (15ml) (Corning)
- Eppendorf tubes (1.5ml) (Eppendorf)
- Acrodisc White Blood Cell filters (25mm diameter) (Cytiva)
- Ice making machine (Kojak)
- Magnetic activated cell sorting (MACS) buffer (Miltenyi Biotech)
- Ficoll paque (Cytiva)
- Swinging Bucket centrifuge (International Equipment Co. A division of Damon)
- CD71 human antibodies (Miltenyi Biotech)
- MACS column (Miltenyi Biotech)
- MACS separator (Miltenyi Biotech)
- Light microscope (Leica microsystems)
- Countess III automated cell counter (Thermo Fisher Scientific)
- Countess cell counting chamber slides (Thermo Fisher Scientific)
- New methylene blue (NMB) (Sigma Aldrich)
- 100- 1000μl Micro pipette (Agros 240-21 Omega 8)
- 0.1- 10μl Micro pipette (Agros 240-21 Omega 8)
- 100μl Pippete tips (Argos technologis)
- 10μl Pippete tips (Argos technologis)
- 1000μl Pippete tips (Argos technologis)
- Glass slides(Corning)
Safety warnings
Time is of utmost importance, as high cell viability is necessary for downstream processing of the enriched reticulocytes for single cell transcriptomics.
Ethics statement
This protocol needs prior approval by the users' institutional review board (IRB) or equivalent ethics committee(s).
Before start
Ensure to have all materials and reagents ready before you begin. This is because reticulocytes continue to mature rapidly into RBCs, which could lead to decrease in their number and significant loss after enrichment.
Protocol
Protocol
Stage 1: Sample collection
Collect 5 mL of blood sample by venepuncture in an EDTA tube.
Immediately place the sample on ice and transport to the lab for further analysis.
Move to stage 2 within two hours of sample collection.
Stage 2: Reticulocyte Enrichment
Aliquot the blood samples, 2 mL each, into separate 15 mL falcon tubes.
Dilute 2 mL blood sample with an equal volume of MACS buffer.
Into another 15ml falcon tube, layer 2 mL of ficoll and then 2 mL of the diluted blood sample above the ficoll.
Centrifuge at 4000rpm for 45mins.
With the help of a micropipette, collect the RBC package (1 mL ) at the bottom of the tube, and transfer into a new 15 mL falcon tube.
Dilute the RBC package with 1 mL of MACS buffer.
Filter the diluted sample through a WBC filter (25mm diameter), to leukodeplete the sample.
Collect the flow-through into a new 15ml falcon tube.
Add 0.1 mL of CD71 antibody to 1ml of the filtered sample and incubate at room temperature for 15 minutes.
Mix the sample gently and put 1 mL of the sample into the MACS column attached to the MACS separator.
As the sample flows through the column, add 1 mL of MACS buffer to wash.
Add another 1 mL of MACS buffer for a second wash.
Add another 1 mL of MACS buffer for a third wash.
Add another 1 mL of MACS buffer for a fourth wash.
Add another 1 mL of MACS buffer for a fifth wash.
Discard the flow-through, which is mostly matured RBCs.
The reticulocytes are retained on the magnetic beads in the MACS column, due to their binding to the CD71 antibodies.
· Elute the CD71+ cells (reticulocytes) using 1 mL MACS buffer, with the help of a plunger, into a new 1.5 mL Eppendorf tube.
The tubes containing the sample should always be on ice.
Move to stage 3 immediately
Stage 3: Visualisation and determination of cell
viability
Into a new 1.5 mL Eppendorf tube, take 10 µL of the CD71+ sample and stain with 10 µL of New methylene blue (NMB).
Incubate for 15min at room temperature and view under the microscope.
Also, take 10 µL of the CD71+ sample and put into a Countess cell counting chamber slide.
Insert the chamber slide into a Countess 3 automated cell counter to count the cells and determine their percentage viability.
The cells are ready to be used for further analysis as required.