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

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Team SUSTC-Shenzhen

Notebook

Elements of the endeavor.

Determining the appropriate concentration of blasticidin for cell selection.

2014/8/27 Prepare for selection

Introduction

The plasmid pBX-083 for the stable transfection of EGFP gene encodes blasticidin resistance. So the minimum level of blasticidin 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 1-3μg/ml (from Invitrogen).

Materials

  • Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1x Penicillin-Streptomycin
  • HeLa cells.
  • Blasticidin(bla) (10mg/ml).
  • 24 well plate.

The design of the experiment:(the concentration of Blasticidin, μg/mL)

The blasticidin gradient
0 μg/ml 0.5 μg/ml 1.2 μg/ml 1.6 μg/ml 2.0 μg/ml 2.4 μg/ml
0 μg/ml 0.5 μg/ml 1.2 μg/ml 1.6 μg/ml 2.0 μg/ml 2.4 μg/ml
2.8 μg/ml 3.2 μg/ml 3.6 μg/ml 4.0 μg/ml 5.0 μg/ml 6.0 μg/ml
2.8 μg/ml 3.2 μg/ml 3.6 μg/ml 4.0 μg/ml 5.0 μg/ml 6.0 μg/ml

Procedures

Aug.27 Seed plate 10,0000 cells per well

  1. Wash cells with PBS, trypsin digestion, add complete medium to stop digestion
  2. Transfer the cell suspension into 15 mL centrifuge tube and centrifuge at 1000rpm for 5 min.
  3. Discard the supernatant and add 2 mL complete medium to suspend the cells.
  4. Dilution and count the cell concentration with blood counting chamber.
  5. Take 2,400,000 cells and add medium to get a total volume of 24mL, mix well.
  6. Add 1mL cell diluted suspension got in step 5 to each well.
  7. Shake the plate to make cell distribution uniform.


(On the evening of 28th Aug) Culture cells in the medium containing blasticidin:

  1. Add 100μL 5mg/mL Blasticidin to 50mL complete medium, mix well to get medium containing 10μg/mL Blasticidin.
  2. Get medium of different concentration of Bla. By adding different volume of medium containing 10μg/mL Blasticidin and corresponding medium without antibiotic. (Eg: 1.6μg/mL = 160μL 10μg/mL + 840μL medium without antibiotic.) Mix well, discard the old medium and add 0.5ml to the well of responding concentration.
  3. Observe the morphology of the cells in the following day. And the replace new medium with corresponding concentration of Blasticidin with the old medium every 2 or 3 days.


Results

31th Aug.

Figure 1.

The concentration blasticidin
No more than 4.0μg/mL No obvious morphology changes
5.0μg/mL Half cells are suspended
6.0μg/mL About 20-30% cells are still adherent

1st Sept.

Figure 2.

4 days blasticindin treatment
<2.4 μg/mL No obvious morphology changes
>=2.4μg/mL Many cells are suspended
>=3.6μg/mL Large amount of cells are dead

2nd Sept.

Figure 3.

3rd Sept.

Figure 4.

4th Sept.

Figure 5.

5th Sept.

Figure 6.

Conclusions

Conclusions:

  • 4.0μg/mL Bla is enough to kill all HeLa cells within 4 days.
  • 3.6μg/mL Bla is enough to kill all HeLa cells within 6 days.
  • 3.2μg/mL Bla is enough to kill all HeLa cells within 7 days.
  • 2.8μg/mL Bla is enough to kill all HeLa cells within 8 days.


Maintained by the iGEM team SUSTC-Shenzhen.

Licensed under CC BY 4.0.

  • ©2014 SUSTC-Shenzhen

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