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Why It Issues and three Simple Strategies

Cell confluency can have an effect on cell habits and progress, so it’s essential to precisely and reliably measure cell confluency. However what’s confluency? How do you measure it? And why is it a vital consideration in your experiments?

What Is Cell Confluency?

An environment friendly cell tradition workflow is significant in lots of analysis areas within the life sciences and the biopharmaceutical business and includes checking cell confluency.

Importantly, cell confluence isn’t the identical as cell quantity. As an alternative, it’s outlined as the share space coated by adherent cells in a tradition dish or flask. (1) This can be a routine measurement used to trace cell proliferation throughout cell tradition.

Cell confluence is essential for figuring out timings for splitting (or passaging) and harvesting cells and for drug remedies or differentiation experiments (Determine 1). Correct and reproducible measurements are very important for producing high-quality, dependable information. In business, correct measurements are particularly essential for standardizing cell tradition protocols for growing and manufacturing cell therapies. (1,2)

How Confluent Are Your Cells? A Beginner's Guide to Measuring Cell Culture Confluency
Determine 1. Typical cell tradition workflow. Cell confluency is checked throughout routine passaging and earlier than experimental use. (Picture Credit score Laura Grassie)

Why Is Cell Confluency Vital?

Excessive ranges of confluency can dramatically have an effect on cell habits and tradition kinetics. (1) For instance, at excessive confluency, myoblasts and preadipocytes can spontaneously differentiate into myotubes and lipid droplet-containing adipocytes, respectively.

Differentiation also can have an effect on the proliferative potential of cultures. Which means that if cells have to be cryopreserved for later use, they need to be frozen at subcritical confluence. This might be 70% for myoblasts, however this worth depends on the cell line. (1)

Impact of Confluency on Cell Well being

Cell loss of life can occur at excessive confluency as a result of vitamins within the media develop into depleted or cells begin competing for area on the tradition dish or flask floor. Due to this fact, if cells are harvested and cryopreserved at a excessive or essential confluence, many and even the entire cells could die as soon as thawed.

If this occurs, you might need to make use of extra of your cell shares, and your cells could take longer to develop and attain the required confluence. Both method, this may trigger large complications and be an enormous waste of your time and assets.

Drug Discovery and Confluency

It’s additionally essential to contemplate cell confluency in cell-based drug discovery initiatives. Some of the essential units of experiments in my PhD relied on utilizing cells at low confluency and treating them with a drug to find out efficacy. It was crucial to make use of cells at low confluency to exclude any non-specific results brought on by over confluent cells and never from the check drug.

Impact on Morphology

Cell confluency can also be an important consideration should you’re learning cell morphology over time. Some cells could have one morphology at low confluence and a special one at excessive confluence. For instance, NIH3T cells are flat and elongated at low confluence, however after they develop into confluent (i.e., 70 – 80% confluence), they re-arrange into an organized brick-like monolayer, which impacts different cell traits. (1)

The way to Decide if Your Cells Are Confluent

Given its significance in cell-based experiments, there are numerous strategies for measuring cell confluency. Nevertheless, at present out there strategies are sometimes subjective, complicated, harmful, and/or time consuming. A few of these strategies are summarized in Desk 1.

Chemical dyes  
(e.g., Thymidine, Alamar blue, XTT and so on.) 

Comparatively quick and may be measured on a plate reader (colorimetric or fluorescent) 
Reasonably priced 

Oblique measure of confluency 
Thymidine causes cell loss of life on account of radioactivity 
Alamar blue and XTT measurements should not absolute and require an ordinary curve to estimate confluency 
Requires manipulation of cultures and is harmful 

Qualitative visible measurement  

Permits for continued undisturbed progress of cells within the tradition disk/flask 
Reasonably priced 

Subjective and might differ relying on the researcher(s) doing the visible inspection 
Can introduce variability between experiments, particularly if completely different researchers are performing the visible verify for one experiment 
Relying on the variety of tradition dishes/flasks that have to be checked, this is usually a time-consuming course of 

Picture processing strategies (e.g., Olympus CKX53 tradition microscope, CKX-CCSW confluency checker software program, and the Air Fraction (AF) output (3) 

Correct, goal, and constant 
Knowledge acquisition and evaluation may be automated  
Comparatively quick 
Permits for continued undisturbed progress of cells within the tradition disk/flask 

Can require costly and specialised microscopy tools and software program algorithms 
Evaluation may be sophisticated 

Desk 1. Strategies for measuring cell confluency.

What Is Meant By the Percentages?

Cell confluency is usually given as a share, which refers back to the proportion of the tradition dish or flask coated by adherent cells (e.g., HEK293 cells). For instance, if cells are round 80% confluent, a monolayer of those cells covers 80% of the floor of the tradition dish or flask. If you’re not skilled in measuring confluency, it may be a troublesome guessing recreation, particularly if you’re utilizing qualitative visible measurements. Under are some easy guidelines and graphical representations that can assist you with this.

50% confluent

This can be a comparatively straightforward estimation as a result of the realm coated by cells ought to be much like the realm not coated by cells (Determine 2).

How Confluent Are Your Cells? A Beginner's Guide to Measuring Cell Culture Confluency
Determine 2. Cells at ~50% confluency cowl half of the floor space.

70% – 80% confluence

At this confluency, cells ought to nonetheless be rising exponentially however nearing the top of log-phase progress. They are going to cowl a lot of the dish, however gaps are nonetheless current (Determine 3).

How Confluent Are Your Cells? A Beginner's Guide to Measuring Cell Culture Confluency
Determine 3. Cells at ~70-80% confluency cowl a lot of the dish, however gaps and areas are nonetheless current.

Ideally, cells ought to be break up at this stage as a result of it will enhance general cell viability. Splitting your cells at this stage may even result in much less aggregated cells. These cells may even have a shorter lag time (i.e., the time taken earlier than cells begin logarithmic progress) after they’re break up and/or thawed after cryopreservation.

100% confluence

Once more, that is one other easy estimation as a result of, on this case, your entire floor of the tradition vessel will probably be coated by cells with no area seen between them (Determine 4). If cells attain this degree of confluence, one in every of two issues will occur:

  1. Regular cells have contact inhibition and can not often attain 100% confluence.
  2. Immortalized cells don’t have contact inhibition, which implies that after they’re 100% confluent, they’ll squeeze collectively, and crowding will trigger cells to scale back to half of their unique dimension. If these cells aren’t break up, they’ll begin detaching from the flask floor, resulting in cell loss of life.
How Confluent Are Your Cells? A Beginner's Guide to Measuring Cell Culture Confluency
Determine 4. Cells at ~100% confluency cowl your entire dish with no gaps current. Cells could also be careworn and bear cell loss of life on account of competitors for assets and overcrowding.

Typically, it is best to keep away from letting your cells develop into over confluent as a result of they’ll doubtless begin to die off (generally in a short time). They will not be recoverable, particularly after being saved in liquid nitrogen for a very long time. Learn our “What’s in a Quantity: Getting the Proper Passage in Cell Tradition” article to be taught extra about how and when to separate your cells.

Instruments for Estimating Confluency Extra Precisely

When you develop into extra skilled at estimating cell confluency by qualitative visible inspection, it’ll develop into simpler, and your measurements will (hopefully) develop into more and more correct. Nonetheless, automated image-based strategies are the gold commonplace for accuracy, reliability, and reproducibility. Examples of such instruments are given beneath.

Air Fraction (AF) Output

This methodology makes use of a easy cardboard coverslip that may be reduce to dimension to suit completely different tradition flasks. Cells are imaged utilizing an inverted phase-contrast gentle microscope, and pictures are captured with a digital digital camera outfitted with a digital camera lens adaptor. Photos can then be analyzed for confluence utilizing the free ImageJ software program. The output is a measure of confluence, known as an Space Fraction (AF). (3)

The Private AUtomated Lab Assistant (PAULA)

The PAULA from Leica Microsystems can be utilized on the bench, within the hood, and even within the incubator, permitting you to check your cells of their pure state. On this method, the cells bear much less stress which is at all times good! The PAULA takes photographs in seconds, permitting you to verify the confluency of a number of flasks of cells in seconds fairly than spending hours at an everyday microscope.

For those who’re learning giant cell populations with completely different fluorescent markers, or should you’re learning morphological modifications over time, the PAULA will probably be helpful for you because it helps multi-fluorescence and time-lapse imaging of cells beneath physiological situations within the incubator.

For added comfort, the PAULA additionally connects wirelessly to any Home windows, iOS, or Android appropriate cellphone or pill, permitting you to verify the standing of your time-lapse in between coffees and experiments.

Olympus CKX53 Tradition Microscope and CKX-CCSW Confluency Checker

Cell confluency may also be precisely measured utilizing a mixture of the CKX53 tradition microscope and CKX-CCSW confluency checker software program. In contrast to with the PAULA, cultures have to be taken out of the incubator for imaging, however this methodology can also be non-destructive, that means there’s no have to take samples out of your tradition flasks.

This helps to protect cells in your downstream work, and this additionally limits the danger of contamination, all crucial when you have a restricted provide of cells in your experiments.

Right here, the CKX53 microscope may be linked to the CKX-CCSW confluency checker software program so you possibly can visually examine your cells after which seize photographs in a single easy workflow. The software program can then precisely and reproducibly calculate cell confluency.

Cell Confluency Summarized

I hope I’ve given you an insightful overview of the significance of precisely measuring cell confluency. I’ve given you some examples of instruments you should utilize to attain this within the lab, however that is in no way an exhaustive checklist. If in case you have any suggestions and tips you’d wish to share, please achieve this within the feedback beneath.


  1. Topman G, Sharabani-Yosef O, Gefen A. (2011) A way for fast, low-cost automated confluency measurements. Microsc Microanal. 17(6):915–22.
  2. Cell Confluency Device. [Accessed 2022 Mar 20].
  3. Non-invasive and non-destructive measurements of confluence in cultured adherent cell traces Elsevier Enhanced Reader. [Accessed 2022 Mar 20].

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