TITLE: Isolating a Monoclonal Cell Population by Limiting Dilution
AUTHORS: Addgene the Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for isolating monoclonal cell population by limiting dilution. To see the full abstract and additional resources, please visit the </span><a href="https://www.addgene.org/protocols/limiting-dilution/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Addgene protocol page</span></a><span style = ":;">.</span></div><div class = "text-block"><span style = "font-weight:bold;">Sample Data</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bkyzn.png" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bkyzw.png" /></div><div class = "text-block"><span style = ":;vertical-align:super;">1</span><a href="https://www.addgene.org/52962/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">lentiCas9-Blast</span></a><span> was a gift from Feng Zhang (Addgene plasmid </span><a href="https://www.addgene.org/52962/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">#52962</span></a><span>) and is described in </span><a href="https://www.ncbi.nlm.nih.gov/pubmed/25075903" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;font-style:italic;">Improved vectors and genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8):783-4.</span></a></div></div>

[STEPS]
?. [Generating Conditioned Medium (optional)]
Seed stable cells such that the next day they will be approximately 50-60% confluent. For Lenti-X 293T cells this is about 2 × 106 cells in a 10 cm dish. Each 10 cm dish should be seeded in  DMEM complete, which will generate enough conditioned medium for one 96-well plate.
10 ml
?. [Generating Conditioned Medium (optional)]
Allow the cells to grow overnight.
?. [Generating Conditioned Medium (optional)]
Replace the medium on the cells and collect the used (conditioned) medium.
?. [Generating Conditioned Medium (optional)]
Remove any cells from the conditioned medium by filtration through a 0.45 µm PES filter or centrifugation at > for .
Do not use the medium if the cells are overly confluent or the medium is acidified (i.e., the medium appears orange/yellow if using a standard phenol red pH indicator) because the buildup of waste products could be suboptimal for cell growth. This conditioned medium will be used to make a cell solution later in this protocol.
?. [Isolating a Monoclonal Cell Population by Limiting Dilution]
Isolate cells from the stable cell pool by trypsinization and break up any cell clumps into individual cells by passing several times through a serological pipet or by passing through a 0.40 µm cell strainer mesh. Quantitate the cell concentration in this homogenized cell solution with a hemocytometer or other cell counter.
*Pro-Tip* Prepare a relatively dilute cell solution (6 cells/mL) before counting the cells. When you ultimately transfer approximately 50-100 cells to the conditioned medium, it will be more accurate if a higher volume of homogenized cell solution is transferred.
?. [Isolating a Monoclonal Cell Population by Limiting Dilution]
Transfer cells from the homogenized cell solution into the conditioned medium prepared above to make a new cell solution at a concentration of 5 cells/mL. Prepare approximately  of this cell solution for each 96-well plate you plan to seed. This 5 cells/mL solution will be used to seed the 96-well plate.
10 ml
If you chose not to use conditioned medium, prepare this cell solution in fresh complete medium.
?. [Isolating a Monoclonal Cell Population by Limiting Dilution]
?. [Isolating a Monoclonal Cell Population by Limiting Dilution]
Transfer  of the 5 cells/mL solution into each well of a 96-well plate. By doing this, you are seeding the plate at an average density of 0.5 cells/well. Seeding an average of 0.5 cells/well ensures that some wells receive a single cell, while minimizing the likelihood that any well receives more than one cell.
100 µl
*Pro-Tip* In each 96-well plate, seed one of the corner wells with ~1000 cells. This will help you focus the microscope later when scanning the plate for individual cells, which can be difficult to find.
?. [Isolating a Monoclonal Cell Population by Limiting Dilution]
Leave the cells undisturbed in an incubator for 7-14 days. After ~7 days, scan the plate for cell growth. If you do not observe any colonies, continue incubating the cells.
*Pro-Tip* Use the well with 1000 cells to focus the microscope, then you can easily scan the plate for individual cells, which should be on the same focal plane.
?. [Isolating a Monoclonal Cell Population by Limiting Dilution]
Scan the entire plate and make note of each well in which you see growth. At this time, the cells will appear as colonies in the well (Figure 1). You will be able to tell if there was more than a single cell seeded in a well because you will observe more than one colony. Wells with more than a single colony do not contain a monoclonal population and should be discarded.
*Pro-Tip* When scanning the plate, be sure to look thoroughly within each well, especially in the corner of the well as cells tend to settle there.
?. [Isolating a Monoclonal Cell Population by Limiting Dilution]
Once the cells have expanded but before they become over-confluent, trypsinize the cells and expand them to larger culture dishes, being sure not to cross contaminate the independent monoclonal lines.
*Pro-Tip* When trypsinizing cells from the 96-well plate, use a microscope to ensure that the cells have detached from the plate and can be isolated. When trypsinized, detached cells will exhibit a rounded morphology.
?. [Isolating a Monoclonal Cell Population by Limiting Dilution]
After the monoclonal lines have been sufficiently expanded, screen the lines for transgene expression and/or other phenotypes. For example, perform Western blotting to screen for lines with the highest or lowest transgene expression (Figure 2).