Flow Cytometry to Identify Bone Marrow Cells

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Flow cytometry is a powerful tool for identifying and analyzing individual cells within a solution. By using lasers and detecting scattered and fluorescent light signals, specific properties like size, granularity, and the presence of certain cell surface markers can be measured, recorded, and analyzed to gain more information about the cells in the sample. To detect the presence of cell markers, fluorescent antibodies that emit light at a certain wavelength can be used. Different varieties of cell markers in a population will create different fluorescent profiles, allowing for the identification of certain types of cells.

For the study of bone marrow cells, the ability to identify cell types is crucial, as bone marrow contains a diverse set of cells, like stem cells, progenitor cells, and mature blood cells. Accurately identifying bone marrow cells is key to diagnosing hematological disorders like leukemia, lymphoma, and myelodysplastic syndromes. Analyzing bone marrow cells through flow cytometry can also aid in stem cell research, immune response research, and assessing treatment effects like the loss of bone marrow cells after chemotherapy. Flow cytometry is invaluable in both research and clinical diagnostics because of its ability to provide rapid, quantitative, and multiparametric analysis of complex bone marrow cell populations.

Intracellular staining of protein biomarkers using antibodies in flow cytometry has opened a diversity of new options for phenotypic and functional characterization of cells in both research and clinical diagnosis. However, definitive phenotypic identification of certain cell types can require labeling both cell surface and intracellular markers. Additionally, the cytoplasmic localization of molecules typically found on the cell membrane can prove to be a reliable lineage marker, such as cytoplasmic CD3 and CD22 in undifferentiated leukemia (1). For even greater cell population stratification, intracellular and intranuclear markers are key, like being able to stain for both proliferation and (anti-)apoptosis markers in hematopoietic cells (2).

FIX&PERM® fixation and permeabilization buffers enhance flow cytometry

To expand the functionality of flow cytometry in research and diagnostic applications, the simultaneous staining of intracellular and cell surface markers is necessary, but not all fixation and permeabilization buffers can handle the task without compromising the quality of your cell samples. Nordic-MUbio and Exalpha’s FIX&PERM® system (GAS-002) utilizes two reagents and a simple protocol to prepare cells for flow cytometry. Reagent A (GAS-002A-1) gently fixes cells, while Reagent B (GAS-002B-1) permeabilizes cells. The FIX&PERM® formulation allows for simultaneous application of the permeabilization medium and antibodies conjugated with fluorescent compounds, enabling the staining of both intracellular and extracellular proteins.

FIX&PERM® buffers contain proven technology, so you know your cells are in good hands. Since the system was first characterized in 1994 by Knapp et al. (3), FIX&PERM® has been independently validated multiple times (4 & 5) and cited in thousands of studies for research on topics such as cancer biology, infectious disease monitoring, and more. Additional advantages include:

• Mild cell fixation, preserving their flow cytometric scatter characteristics
• Rapid technique – whole procedure can be carried out in less than one hour, ready for immediate analysis or storage for 24 hours
• Stringent QC procedures – the quality of each lot is confirmed using well-defined blood samples and subsequent comparison of scatter characteristics and immunostaining patterns, ensuring consistent and reliable results each time
• Stable – formulations do not contain volatile organic solvents, enabling storage at ambient temperature during shipment and in laboratories
• Flexible – can be used with most monoclonal antibody conjugates
• Simple protocol, starting with 50 µl of whole blood, bone marrow or mononuclear cell suspension
• CE/IVD registered kits available for European customers in a clinical laboratory setting

Recombinant antibodies for precise cell marker detection

Successful flow cytometry staining also requires using the right antibodies for the task, but not all antibodies are created equal. Mitigating nonspecific binding begins at the antibody selection stage, and recombinant monoclonal antibodies offer 100% biological definition to ensure specific binding and engineering options for further experimental capabilities.

Here are recombinant monoclonal antibodies from Absolute Antibody that target common intracellular and cell surface markers for bone marrow cell identification and analysis.

• CD3 epsilon: This is a T cell marker usually found on the surface but can help diagnose undifferentiated leukemia when found in the cytoplasm (1). The recombinant anti-CD3 epsilon antibody clone UCHT1 (Ab00112) can be used to stain peripheral blood mononuclear cells (PBMCs) for flow cytometric analysis.

• CD20: This is a mature B cell marker and higher concentrations than normal can signal certain types of B-cell lymphomas and leukemias. Our recombinant anti-CD20 antibody (Ab00126) is the same sequence as the clone 10F38 (rituximab) which is used to detect CD20 in flow cytometry and as a therapy to enhance immune response to cancerous cells.
• CD4: This molecule is part of the immunoglobulin super-family, expressed on the surface of helper T cells. Our recombinant version of the anti-CD4 antibody (Ab00450) is reactive with chimpanzee and human species and can be used for the flow cytometric identification of T cells and certain early hematopoietic progenitor cell.
• CD41 (GPIIb/IIIa): CD41 is a marker for megakaryocytes and platelets. Our recombinant anti-CD41 clone 7E3 (Ab00282) can be used in flow cytometry to identify megakaryocyte progenitors and mature megakaryocytes, as well as monitor disorders related to platelet and megakaryocyte production like myeloproliferative neoplasms.

These antibodies are stored in PBS solution without sodium azide, meaning nothing in our antibody solutions will adversely affect or degrade your cells.

Conclusion

Bone marrow cells are a diverse group of cell types that are the basis of a variety of processes and pathologies. High-quality reagents, including FIX&PERM® cell buffers and recombinant engineered monoclonal antibodies, can ensure your cells stay intact throughout your experiments and keep nonspecific binding to a minimum to power accurate flow cytometric identification and analysis of bone marrow cells.

References

1. Janossy, G., Coustan-Smith, E., & Campana, D. (1989). The reliability of cytoplasmic CD3 and CD22 antigen expression in the immunodiagnosis of acute leukemia: a study of 500 cases. Leukemia, 3(3), 170–181.
2. Mestrum, S. G. C., Vanblarcum, R. B. Y., Drent, R. J. M., Boonen, B. T., van Hemert, W. L. W., Ramaekers, F. C. S., Hopman, A. H. N., & Leers, M. P. G. (2022). Proliferative and anti-apoptotic fractions in maturing hematopoietic cell lineages and their role in homeostasis of normal bone marrow. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 101(7), 552–563.
3. Knapp, W., Strobl, H., & Majdic, O. (1994). Flow cytometric analysis of cell-surface and intracellular antigens in leukemia diagnosis. Cytometry, 18(4), 187–198.
4. Groeneveld, K., te Marvelde, J. G., van den Beemd, M. W., Hooijkaas, H., & van Dongen, J. J. (1996). Flow cytometric detection of intracellular antigens for immunophenotyping of normal and malignant leukocytes. Leukemia, 10(8), 1383–1389.
5. Lanza, F., Latorraca, A., Moretti, S., Castagnari, B., Ferrari, L., & Castoldi, G. (1997). Comparative analysis of different permeabilization methods for the flow cytometry measurement of cytoplasmic myeloperoxidase and lysozyme in normal and leukemic cells. Cytometry, 30(3), 134–144.