Quantification of cell proliferation using WST-1 reagent on 3D scaffolds

Instruments and reagents

•  Bio-Rad Benchmark Plus Microplate Reader (Bio-Rad Laboratories Inc., USA) with computer
•  96 Well Plates
•  WST-1 reagent (Roche, USA)
•  Incubator
•  Cell counting chamber
•  Centrifuge
•  Cell culture materials (Cells, Dishes, Media, PBS and so on)

Protocol

-  Thawing cells and proliferate confluent cell monolayer for cell culture experiment.
-  Sterilize a scaffold with 70% ethanol for 30 minutes.
-  Rinse a scaffold with PBS 3 changes.
-  Exchange PBS to cell culture media and then keep it in an incubator for 24 hours.

1.  Seed cells (1.0-3.0x10^6 cells/scaffold∙ml) in scaffold (diameter: 16 mm)-microplates (24 wells) in a final volume of 1000 ㎕/well culture media in a incubator (37℃, 5% CO2). Incubate for 4 hours, then move scaffolds to a 100 mm new dishes filled with fresh media respectively. Culture cells (day 1, 3, 5) without media exchange.

2.  Prepare 24 well plates fill with new media (1000㎕/well) and blank media before 1 hour.

3.  Take out WST-1 reagent.

4.  Turn on a microplate reader.

5.  Incubate for 60 minutes in an incubator.

6.  Add 100 ㎕/well of WST-1 reagent. Shake a microplate by hands carefully.

7.  Incubate the cells for 15 minutes in an incubator. Check a WST-1 reagent and shake again and incubate 15 or 45 minutes carefully.

8.  Remove WST-1 reacted scaffolds from a 24 well plate quickly.

9.  Move WST-1 reacted solution (100 ㎕) to a 96 well plate for a microplate reader-assay using a 200 ㎕ pipette.

10.  Open the cap of a 96 well plate, and measure the absorbance of the samples against a background control as blank using a microplate reader at 420-480 nm (Max. 440 nm, endpoint protocol). The reference wavelength>600 nm (we use 440 and 650 nm)

Working concentration of WST-1 reagent
10 v/v% of original cell culture medium volume.

 Incubation period of WST-1 reagent with cells

30 or 60 minutes with 1.0-3.0 x 10^6 cells/well∙scaffold∙ml (24 wells plate). It depends on cell concentration, experimental condition and limited absorbance intensity.

Blank for background absorbance
Same volume of culture media + same volume (10 v/v%) of WST-1 reagent.

[References]
http://www.roche-applied-science.com/support

[Remarks]
-  Media의 양을 모든 실험에서 동일하게 할 것
-  블랜크를 만들 때, 동일한 양을 사용할 것
-  WST-1의 반응시간을 정확히 지킬 것
-  마지막에 잘 섞어주고, 스케폴드에 묻어있는 반응물도 모두 짜낼 것
-  WST-1시약은 미리 분배해놓은, 냉동고에서 바로 꺼내어 상온에서 녹여 쓸 것
-  처음 콘트롤용 샘플을 준비시, 스케폴드 콘트롤과, 2D 모노레이어 콘트롤도 제조할 것. 즉, 그냥 웰 플레이트에 동일한 양의 세포를 분주, 배양한 것.
-  3D 스케폴드에서 세포배양시, 세포의 양은 매우매우매우 중요하다. 왜냐하면 2D와3D는 세포의 밀도나. 세포간의 상호작용, 상호정보전달이 다르기 때문이다.