How many cells are in 1ml PBMC?

Peripheral blood mononuclear cells (PBMCs) are an important cell population in immunological research. They consist of lymphocytes (T cells, B cells and NK cells) and monocytes. When isolating PBMCs from whole blood, it is useful to know the approximate number of cells that can be obtained per ml of blood. This allows researchers to plan experiments appropriately.

What are PBMCs?

PBMC stands for peripheral blood mononuclear cell. As the name suggests, these are mononuclear cells found in peripheral blood. Mononuclear cells have a round, single nucleus. Examples of mononuclear cells include lymphocytes and monocytes.

Lymphocytes are a type of white blood cell that are critical for adaptive immune responses. The main types of lymphocytes are:

– T cells: Orchestrate cell-mediated immunity. Recognize antigens on infected or cancerous cells.

– B cells: Generate antibodies. Recognize extracellular pathogens and toxins.

– Natural killer (NK) cells: Mediate innate immunity against viruses and cancerous cells.

Monocytes are white blood cells that can differentiate into macrophages and dendritic cells. Macrophages are phagocytic cells that ingest and destroy pathogens, dead cells, and debris. Dendritic cells act as antigen presenting cells that activate T cells.

Together, lymphocytes and monocytes make up the mononuclear cell population in peripheral blood. That is why they are referred to as peripheral blood mononuclear cells or PBMCs. These are the mononuclear immune cells circulating in the bloodstream.

PBMC isolation

PBMCs are commonly isolated from whole blood using density gradient centrifugation. An anticoagulant such as EDTA is added to the blood sample to prevent clotting. The blood is layered over a density gradient medium such as Ficoll-Paque. This medium has a density between that of PBMCs and other blood components.

When the sample is centrifuged, the cells separate out into layers based on their density. The granulocytes and erythrocytes get pelleted at the bottom. A buffy coat layer containing PBMCs and platelets forms above the erythrocyte layer. The PBMCs can then be collected from this interface.

Isolating PBMCs in this way provides a purified, functional population of mononuclear cells for downstream immunological assays. Common assays performed on PBMCs include:

– Proliferation assays
– Cytokine production assays
– Immunophenotyping by flow cytometry
– DNA/RNA studies

Knowing the number of cells in a PBMC preparation is important for optimizing cell-based experiments. Next, we will discuss how to determine the cell count.

Counting cells in PBMC samples

To determine the concentration of cells in a PBMC sample, an aliquot is taken and counted manually or using an automated cell counter. PBMC counts are usually expressed as the number of cells per microliter (cells/μl).

Manual counting

A hemocytometer is a specialized slide with etched grids that allows manual counting of cells suspended in liquid. Here is the basic protocol:

1. Mix PBMC sample gently to obtain an even suspension.

2. Pipette 10 μl of well-mixed cell suspension into each chamber of the hemocytometer.

3. Place slide under a light microscope and focus. Count viable cells in the corner squares. Dead cells with stained nuclei are excluded.

4. Calculate the concentration using this formula:

Cells/μl = Average count per square x Dilution factor x 104


– Average count per square = Total cells counted / Number of squares counted

– Dilution factor = Any dilution of the original sample before loading onto hemocytometer

– 104 = Conversion factor to account for chamber dimensions

Manual counting allows visualization of the cells and assessment of viability. But it can be tedious for large sample numbers.

Automated cell counters

Automated cell counters streamline PBMC counting and sizing. They use electrical impedance, optical fluorescence, or flow cytometry principles to quantify cell numbers and viability within minutes.

Many counter models can process raw PBMC samples without any dilution needed. The instrument software automatically calculates the cell concentration in cells/μl.

Automated counters improve efficiency and precision of PBMC counting. But the equipment costs more compared to manual hemocytometers.

PBMC concentration range

Now that we have covered techniques to quantify PBMCs, what is the typical cell concentration?

The number of PBMCs isolated from whole blood depends on factors like:

– Blood volume drawn
– Donor health status
– Processing protocol

But generally, the concentration falls in the range of 1-5 million PBMCs per ml of whole blood.

According to published studies analyzing PBMC yields:

– Healthy donors yield 1-3 million PBMCs per ml blood [1].

– Higher concentrations around 4-5 million/ml are possible with optimized isolation protocols [2].

– Diseases like HIV and diabetes are associated with 25-35% lower PBMC counts compared to healthy controls [3,4].

So for a standard blood draw volume of 8-10 ml from a healthy donor, researchers can expect ~10 million PBMCs in 1 ml of isolated cells. The exact recovery should be quantified by cell counting as described earlier.

Factors affecting PBMC numbers

Why does PBMC recovery vary between individuals and experiments? Some key factors are discussed below:

Blood volume

The number of PBMCs collected is directly proportional to the starting blood volume. Smaller blood volumes like 2-4 ml will yield fewer cells.

Drawing larger volumes increases the total PBMCs. But avoiding excessive volumes reduces donor discomfort. 10 ml is generally sufficient for PBMC isolation [5].


Clotting must be prevented for successful PBMC isolation. EDTA and heparin are commonly used anticoagulants.

Heparin is preferred over EDTA for functional assays since EDTA can impact cell activation and viability [6]. But EDTA gives higher PBMC yields than heparinized blood from the same donor [7].

Density gradient medium

The type and density of gradient medium impacts PBMC separation. Ficoll-Paque and Lymphoprep are well-established, commercial density media for PBMC isolation.

Researchers can also prepare custom gradients, but commercial media offers convenience and consistent separations.

Centrifugation parameters

Centrifugation time, speed and acceleration/deceleration settings affect separation efficiency. Lower speeds may retain platelet contamination, while excessive speeds can reduce viability.

400-800 x g for 15-30 minutes is an effective centrifugation range for routine PBMC isolation [8].

Technician skill

Careful density gradient layering, centrifugation, and extraction of the PBMC interface layer requires developed manual skills. Variability between technicians can contribute to differences in PBMC yield.

Donor characteristics

The donor’s general health, age, genetics and other traits influence PBMC counts. For example:

– Infants have lower lymphocyte counts than adults [9].

– Older individuals tend to have fewer naïve T cells [10].

– Obese subjects may have elevated leukocyte counts [11].

– Ethnicity impacts baseline immune cell frequencies [12].

Where possible, matching donor groups by age, gender, BMI and ethnicity helps control for variability.

Typical PBMC experiments

Now that a suitable number of PBMCs have been isolated, what are some common experiments done with these cells?

Proliferation assays

PBMC proliferation indicates expansion and activation of antigen-specific lymphocytes. Proliferation is quantified by measuring DNA synthesis via:

– 3H-thymidine incorporation
– Ki67 intracellular staining

By stimulating PBMCs with mitogens or specific antigens, researchers can assess the responsiveness of T and B cell subsets.

Around 200,000 PBMCs per well are typically used for proliferation assays in 96-well plates [13].

Cytokine assays

Activated PBMCs secrete cytokines like interferon-gamma and interleukin-2. Measuring cytokine levels informs about T cell function.

Cytokine production is evaluated by:

– ELISA on PBMC culture supernatants
– Intracellular cytokine staining by flow cytometry
– qPCR to assess cytokine gene expression

500,000 – 1 million PBMCs per well are commonly used for cytokine analyses in 24-well plates [14].


PBMCs contain various lymphocyte populations that can be quantified by flow cytometry using fluorescent antibodies.

Key lymphocyte subsets analyzed include:

– CD3+ T cells
– CD4+ helper T cells
– CD8+ cytotoxic T cells
– CD19+ B cells
– CD56+ NK cells

Around 100,000 PBMCs are typically stained per tube for flow cytometry [15]. Multicolor panels allow simultaneous interrogation of multiple cell subsets.

Cell mixes for co-cultures

Combining isolated PBMCs with other cell types creates co-cultures that model immune interactions. Common examples include:

– PBMCs + tumor cells (to assess tumor-specific immunity)

– PBMCs + dendritic cells (to study T cell activation)

– PBMCs + B cells (to provide T cell help for antibody production)

PBMC number is optimized based on the assay. 500,000-1 million PBMCs permit robust lymphocyte responses.


To summarize key points:

– PBMCs are lymphocytes and monocytes isolated from peripheral blood.

– Typical concentrations are 1-5 million PBMCs per ml of whole blood.

– Cell counting by hemocytometer or automated methods determines the concentration.

– Blood volume, donor health, processing protocol and technician expertise impact PBMC yield.

– Quantifying PBMCs is necessary for functional assays like proliferation, cytokine production, immunophenotyping and co-cultures.

– Adjusting input PBMC number based on the assay and well size gives optimal results.

Understanding PBMC quantity allows researchers to design reproducible immunology experiments and interpret results appropriately.

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