TITLE: Monoclonal Culture (Limiting Dilution) 
AUTHORS: Binghua Darren Zhang

[DESCRIPTION]
This protocol gives a step-by-step guidance on how to generate the homogenised gene-knockout (KO) monoclonal cell line from a polyclonal "KO" cells pool. 


[BEFORE_START]
Generate and prepare the stable polyclonal cell pool; 

[GUIDELINES]
Prepare conditioned growth medium;  
Cell counting and calculation; 
Culture single cell suspension in 96-well cell culture plates; 


[STEPS]
SECTION: Prep Conditioned Medium
1. Collect the used growth medium into a 50 mL Falcon tube. 
 


SECTION: Prep Conditioned Medium
2. Pipette the used growth medium through a 0.2 µm syringe filter to remove the waste product and dead cells. 

SECTION: Prep Conditioned Medium
4. Add fresh growth medium up to 30 mL (if there are three 96-well cell culture plates). 30 mL 
 


SECTION: Prep Conditioned Medium
3. Collect the supernatant (about 15 mL) used growth medium into a new 50 mL Falcon tube. 
And then discard the waste product. 15 mL 
 


SECTION: Prep Conditioned Medium
5. Mix the medium thoroughly and seal the tube tightly for later use as the conditioned medium. 

SECTION: Prep the &quot;KO&quot; Cells
6. Rinse cells with sufficient amount of DPBS Buffer. 
15 mL
SECTION: Prep the &quot;KO&quot; Cells
7. Aspirate the DPBS with aspirator. 
SECTION: Prep the &quot;KO&quot; Cells
8. Trypsinise the cells with 1 mL of Trypsin in the culture incubator at 37℃ for 2 min.   1 mL 2 min 

SECTION: Prep the &quot;KO&quot; Cells
9. Add 5 mL fresh growth medium to stop trypsinisation. 5 mL
Mix well. 
SECTION: Prep the &quot;KO&quot; Cells
10. Transport into a new 50 mL Falcon tube and spin at 1,000 rpm, 5 min. 1000 rpm, 5 min 

SECTION: Prep the &quot;KO&quot; Cells
11. Discard the supernatant as possible using the aspirator. 
SECTION: Prep the &quot;KO&quot; Cells
12. Resuspend the cell pellet with 4 or 5 mL fresh growth medium. 4 mL 5 mL 

SECTION: Prep the &quot;KO&quot; Cells
13. Passage/Subculture cells at a ratio of 1:8 or 1:10 as usual. 
Refer to the Cell Subculture protocol. 
SECTION: Prep the &quot;KO&quot; Cells
14. Pass the rest cell suspension into a new 50 mL Falcon tube through a 0.40 µm cell strainer mesh to break up cell clumps into individual cells.  
SECTION: Calculation
15. Pipette up and down couple times to make sure the cell suspension is evenly distributed. 
SECTION: Calculation
16. Quantitate cell concentration using a hemocytometer. 
 

SECTION: Calculation
17. Sample Calculation (Example Calculation)
SECTION: Calculation
17.1. Expect to have a cell concentration of ~3x106 cells/mL. 
SECTION: Calculation
17.2. 10X Dilution to make the concentration of ~3x105 cells/mL. 
Count cells in replicates. 
SECTION: Calculation
17.3. Expect to have the final concentration at ~5 cells/mL. 
For 30mL conditioned medium, a total of ~150 cells are expected. 
Thus, (~150 cells)/(~3x105 cells/mL) ≈ 0.5 µL of cell suspension is required. 
SECTION: Calculation
17.4. Further dilute cell suspension in 1:100 DF to obtain an expected concentration of ~3x103 cells/mL. 
(~150 cells)/(~3x103 cells/mL) ≈ 50 µL of cell suspension is required. 
SECTION: Monoclonal Culture Plate
18. Pipette 50 µL which carries ~150 cells from the cell suspension into the prepared 30 mL conditioned medium (Section 1). 
Mix thoroughly. 
SECTION: Monoclonal Culture Plate
19. Transfer the medium into a tray. 
SECTION: Monoclonal Culture Plate
20. Use multi-channel pipette to transfer 100 µL into each well, leading to the cell concentration of ~0.5 cell/well. 100 µL 

SECTION: Monoclonal Culture Plate
21. Place one control well containing ~1,000 cells in each plate. 
In this case, pipette ~3 µL from the cell suspension of ~3x105 cells/mL (Section 17.2) into the control well and labelled. 
SECTION: Monoclonal Culture Plate
22. Place the plates into the incubator until they are ready for single cell colonies screening.