Team:SUSTC-Shenzhen/Notebook/HeLaCell/Determining-the-appropriate-concentration-of-puromycin-for-cell-selection

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       title=Notebook|
       subtitle=Elements of the endeavor.}}

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       name=Determining the appropriate concentration of puromycin for cell selection|
       date=2014/8/17|
       goal=Prepare for selection}}
==Introduction==
The plasmid for the stable transfection of Cas9 gene encodes puromycin resistance. So the minimum level of puromycin to be added to the culture medium to prevent the normal HeLa cells growth needs to be determined for the following cell line selection. The recommended concentration of mammalian cell lines is 1-10μg/ml. And for HeLa cell it is 3μg/ml (from Antibiotics gene transfection) and 2μg/ml (from Invitrogen).

==Materials==
* HeLa cells.
* Puromycin (10mg/ml).
* Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS).
* 24 well plate.

==Procedures==
===8.17. Morning===

Preparation for the test of puromycin concentration.
Seed cells on a 24-well plate, seed 100,000 cells in every well and 12 wells in total.

===8.18 Morning===
The cells grow well and uniformly, the confluence was almost 100% before adding medium with puromycin, a small ratio of cells turn round (It might result from the extremely high confluence).
<html><div class="table-responsive"></html>
{| class="table"
! 0ng/μL
! 1 ng/μL
! 1.5 ng/μL
! 2 ng/μL
! 2.5 ng/μL
! 3 ng/μL
|-
| 4 ng/μL
| 1 ng/μL
| 1.5 ng/μL
| 2 ng/μL
| 2.5 ng/μL
| 3 ng/μL
|}
<html></div></html>
Table 1. The puromycin gradient

# Add 0.5mL of complete medium with different puromycin concentration according to the table above.
# 10 h after adding puromycin, there was no obvious change of the morphology of the cells.
#30 h after adding puromycin, the medium was discarded and the cells were washed with PBS and added new medium, and the cells were observed again.

The control group growed well. The higher the concentration of the puromycin, the less living cells could be observed. It is obvious that the most cell in 3 or 4ng/μL puromycin died.

{{SUSTC-Image|wiki/images/4/41/SUSTC-Shenzhen-Puromycin-1.PNG|
Fig 1. The HeLa cells after 30 hours puromycin treatment. The floccules are suspended dead cells and some of them are result from the high cell confluence. The medium was discarded and the cells were washed with PBS, added new medium and then observed under a microscope (4X10X). (the unit of the concentration of the puromycin labeled is μg/mL). The higher the concentration of the puromycin, the less living cells could be observed.}}


===8.20===
{| class="table"
|-
!The concentration of puromycin(μg/mL)
!Description
|-
| 
0
| 
The confluence is very high, and part of cells become round
|-
|1
|High confluence, part of cells are suspended
|-
|1.5
|Many cells are suspended
|-
|2-2.5
|Most cells are suspended
|-
|3-4
|Almost all of the cells are dead
|}

Table 2. The observation after 2-day’s puromycin treatment

{{SUSTC-Image|wiki/images/1/1e/SUSTC-Shenzhen-Puromycin-2.PNG|
Fig 2. The HeLa cells after 2-day’s puromycin treatment. The floccules are suspended dead cells and some of them are result from the high cell confluence. The medium was discarded and the cells were washed with PBS, added new medium and then observed under a microscope (4X10X). (the unit of the concentration of the puromycin labeled is μg/mL).}}

===8.21===


{| class="table"
! The concentration of puromycin(μg/mL)
! Description
|-
| 0
| The confluence is very high, and part of cells become round
|-
| 1
| High confluence
|-
| 1.5
| There are still some living cells
|-
| 2-4
| Few living cells can be found
|}
Table 3. The observation after 3-day’s puromycin treatment
 
{{SUSTC-Image|wiki/images/7/71/SUSTC-Shenzhen-Puromycin-3.PNG|
Fig 3. The HeLa cells after 3-day’s puromycin treatment. The medium was discarded and the cells were washed
with PBS, added new medium and then observed under a microscope (4X10X). (The unit of the concentration of the puromycin labeled is μg/mL).
}}
===8.22 AM===

{| class="table"
! The concentration of puromycin(μg/mL)
! Description
|-
| 0
| The confluence is very high, and part of cells are suspended
|-
| 1-1.5
| Many cells are suspended
|-
| 2- 4
| Almost all of the cells are suspended
|}
Table4. The observation after 4-day’s puromycin treatment

{{SUSTC-Image|wiki/images/3/3e/SUSTC-Shenzhen-Puromycin-4.PNG|
Fig 4. The HeLa cells after 4-day’s puromycin treatment.(4X10X) The floccules are suspended dead cells and
some of them are result from the high cell confluence.(The unit of the concentration of the puromycin labeled is μg/mL).
}}

==The result==

 
# 2.0 μg/mL puromycin is enough to kill all of the normal HeLa cells within 4 days
# 3.0 μg/mL puromycin is enough to kill almost all of the normal HeLa cells within 2 days

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