Measuring Cells

Measurement of cell mass

As its name suggests, cell mass refers to the mass of a single cell or group of cells. Cell mass measurement is not as simple as placing your sample on a normal weighing scale because living cells lie within the picogram range. Instead, researchers use a specialised picobalance that uses a cantilever, a structure containing a horizontal beam or element supported only on one end. By attaching your cells to one end, you can quantify the mass change and calculate the cell mass.

Measurement of cell confluency

Visual estimations prove subjective and will inherently introduce variability. This method includes taking an image of your cell sample and estimating how many cells cover a given area. However, measuring confluency by automated means is the preferred option by scientists, as this method returns accurate data and is quicker than visual estimates. Machines such as Solentim Cell Metric can determine confluence by taking an image of your sample and analysing the area of adherent cells.

Fig. 1 - Four different confluency of cells under the microscope

Measurement of cell size

Cell size measurement is often performed using a graticule (also called a reticule), which contains a scale with 100 divisions on the eyepiece of a light microscope. A stage micrometre, a slide containing a scale of a known length, must be used to calculate how many micrometres are contained within one division on the graticule. Once you calculate this, you can measure the size of any cell.

Calibrating the graticule

As mentioned above, you need a stage micrometre to calibrate your graticule. Outlined below are the steps:

1. Select the lowest magnification on your microscope.
2. Position a stage micrometre on the microscope stage.
3. Position a graticule in the microscope eyepiece such that its divisions align with the stage micrometre scale (it is helpful to align the zeros together).
4. Count the number of divisions on the graticule that align with the stage micrometre.
5. Calculate the micrometres of one division on the graticule.

Fig. 2 - Three fields of view are seen in this figure. A) An eyepiece graticule B) Superimposed cells and an eyepiece graticule C) A micrometre and an eyepiece graticule

Measurement of red blood cell volume

Your blood contains many other cells and components alongside red blood cells. These components include platelets, leukocytes and plasma. Red blood cell volume describes the ratio or percentage of red blood cells to the volume of whole blood; this is most commonly known as haematocrit. In healthy adults, this value should range from 40 to 48%.

Fig. 3 - Separated content of a blood sample

Patients will have their blood drawn in clinics and hospitals, and automated haematology analysers will return the haematocrit count.

A low haematocrit count indicates an abnormally low percentage of red blood cells and that the patient may be experiencing:

• Haemorrhage
• Anaemia
• Bone marrow defects

A high haematocrit indicates an abnormally high percentage of red blood cells and that the patient may be experiencing:

• Dehydration
• Polycythaemia vera

Measurement of cell migration

Cell migration describes the rate of cell motility. This measurement is key as motility is involved in important processes such as tissue development and immune responses. There are different types of assays that researchers use to measure cell migration, such as:

• Pipette scratch assays (also called the wound closure assay)
• Transwell invasion assays (also called the modified Boyden chamber assay)
• Live imaging

Here, we will focus on the pipette scratch assay and transwell assay.

The pipette scratch assay involves manually creating a wound with a pipette in cell culture. The cells found on the edge of the new wound will migrate towards the cell-free gap. Images are taken at regular intervals throughout the assay duration to calculate the rate of cell migration.

The transwell invasion assay involves a chamber containing an upper and lower layer separated by a permeable layer coated with extracellular matrix proteins, such as collagen. Cells are seeded ("planted") onto the upper layer while the lower layer is filled with media containing a chemoattractant. Over time, the cells will migrate through the membrane, and the rate of cell migration is calculated by counting the migrated cells with a plate reader.

Measurement of cell density

A microfluidic mass sensor (MMS) measures cell density, also termed a suspended microchannel resonator (SMR). This machine can hold two fluids of different densities; one fluid being the cells under investigation and the other fluid being denser. The fluid density and buoyant cell mass are calculated, which allows the machine to calculate single-cell density.

Measuring cell metabolism

Cellular metabolism refers to the sum of all biochemical processes that occur in a cell, including catabolic and anabolic reactions. These processes include cellular respiration, protein synthesis and cellular trafficking. In particular, metabolic reactions change and deviate from the norm in disease states. As you can perhaps imagine, many reactions occur simultaneously; we will take a look at a couple of examples.

Proinsulin metabolism

Proinsulin is the precursor molecule of the more well-known hormone, insulin. Proinsulin is split at two sites to give rise to insulin, which regulates the cellular uptake of glucose. Only trace amounts of unsplit proinsulin are released into the bloodstream in healthy individuals. However, researchers have found that high levels of circulating unsplit proinsulin indicate insulin resistance, specifically in type-II diabetes.

Enzyme-linked immunosorbent assay (ELISA) kits measure the levels of unsplit proinsulin in clinical samples. These assays use antibodies and fluorescence-based technologies to quantify the level of proteins in a sample. By using these kits, you can measure the levels of circulating proinsulin to monitor proinsulin metabolism.

Oxygen consumption

Oxygen is a key substrate in aerobic respiration, and the rate of oxygen consumption is a great indicator of a cell's state. For example, a low oxygen consumption rate may indicate mitochondrial dysfunction. In laboratories, researchers use automated machines to calculate the oxygen consumption rate.