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Cell Starvation Needy Materials: SFM, Pbs (-MGCL Steps

The document provides instructions for two cell biology experiments: cell starvation and comet assay. 1) The cell starvation experiment involves warming media in a water bath, washing cells with PBS, treating cells with BK and H2O2 at different concentrations for periods of time, then changing media and incubating for 18-24 hours. 2) The comet assay involves preparing agarose, lysing and scraping cells, mixing cells with agarose on slides, lysing and treating slides, electrophoresis, staining with SYBR Green, and analyzing results under a microscope. Precise steps and materials are outlined for both experiments.

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Shuying Wu
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60 views5 pages

Cell Starvation Needy Materials: SFM, Pbs (-MGCL Steps

The document provides instructions for two cell biology experiments: cell starvation and comet assay. 1) The cell starvation experiment involves warming media in a water bath, washing cells with PBS, treating cells with BK and H2O2 at different concentrations for periods of time, then changing media and incubating for 18-24 hours. 2) The comet assay involves preparing agarose, lysing and scraping cells, mixing cells with agarose on slides, lysing and treating slides, electrophoresis, staining with SYBR Green, and analyzing results under a microscope. Precise steps and materials are outlined for both experiments.

Uploaded by

Shuying Wu
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOC, PDF, TXT or read online on Scribd
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Cell starvation

Needy materials: SFM, PBS (-MgCl2/-CaCl2), BK, H2O2.

Steps:
1. Warm SFM and PBS (-MgCl2/-CaCl2) in the 37°C water bath about 20 minutes.

2. Remove the 6-well-plates’medium and wash wells by PBS 1 time.

3. Add SFM medium 2ml/well. Put plates into the incubator.

4. After 8hrs add BK (10-7) into the BK groups, 20 µl /well, gently shake the plates then
put into the incubator again. 15 minutes later add different concentrations’ H2O2 into
wells 20µl/well. Gently shaking the plates then put into the incubator.

5. 1h later changes the SFM into DMEM 2ml/well. Put into the incubator
about18~24hrs.
Comet assay

Needy materials: LMAgarose, lysis solution, 1×TBE solution, cell scraper, cell slides, ice,
Steps:

1. Heating agarose (0.7%) in the boiled water about 30 min until it melts then put into
37C water bath 20~30 min. Cool down the centrifuge machine to 4 °C.

2. Per chill standby lysis solution into 4°C refrigerator at least 30min.

3. Mark the slides.

4. Add 900 µl agarose into each tube then put it into 37°C water bath.

5. Remove 2ml/well medium from 6-well-plates.

6. Observe cells quickly before scrape it.

7. Scrape cells gently and pay attention to the top and bottom of the wells, after scrape
the cells observe cell under microscopy to have a check.

8. Collect 2ml cells add into each tube.

9. Counting each groups cell number.

10. Calculate the cell concentration and how many PBS is needed to make sure the cells
concentration is 10×104/ml.

11. Spin down cells in 4°C, 1200rpm, and 8 min then carefully remove the media. Mix the
cells with the left media. Add PBS washing the cells only once by centrifuge in 4°C,
1200rpm, 8 min. Then remove the PBS.

12. Add a specified volume PBS to each tube to make sure the concentration of cells are
10×104/ml.

13. Get out of 70µl cells from each tube add into the standby agarose tubes mix it.

14. Per warm slides in the incubator, add the mixture 70µl onto each slides’ circle; use the
pipette to flatten the circle area carefully.

15. Put the slides into the 4°C situation 20min.

16. Immerse slides into per chilled lysis solution in 4°C, dark, and 1h.
17. Gently drain excess buffer from slides, immerse it into freshly AUS 30min, room
temperature in dark.

AUS (300mM NaOH, 1mM EDTA) Room temperature. ----200ml

NaOH 2.4g
200Mm EDTA 1ml
dH2O 199ml

18. Gently drain excess buffer from slides, immerse it into 1×TBE solution 5min, RT in
dark.

19. Put slides immerse 1×TBE solution 20V, 10 min(1×TBE solution do not exceed 0.5cm
above slides)

20. Gently drain excess buffer from slides, immerse it into dH2O for 5 min.

21. Immerse slides into 70% ethanol for 5 min.

22. Dry samples overnight in dark.


2011-04-18 Staining cell

Needy materials: SYBR Green I (1 µl) + TE buffer (10ml)

--TE buffer Prepare:


--10 mM Tris-Cl, pH 7.5
--1 mM EDTA

Make from 1M stock of Tris-Cl (pH 7.5) and 500 mM stock of EDTA (pH 8.0).
10ml 1M Tris-Cl pH 7.5 per L
2ml 500mM EDTA pH 8.0

1M Tris (crystallized free base)


Tris (hydroxymethyl) aminomethane
FW 121.4 g/mol
60.57 g in 0.5L mq water
pH to 7.5 using HCl

0.5M EDTA
Diaminoethane tetraacetic acid
FW 372.2 g/mol
18.6 g in 100ml mq water
pH to 8.0 using NaOH
• EDTA will not be soluble until pH reaches 8.0
• Use vigorous stirring, moderate heat (if desired) and time

Steps:

1. Place 100µl of diluted SYBR Green I onto each sample, place in refrigerator for 5
min.

2. Gently tap slide to remove excess SYBR solution allow slides to dry completely at
room temperature in the dark.

3. View slides by epiflourescence microscope.

3. Recording the data and analyze it.


Prepare For 50ml DMEM

DMEM 44.5ml
10%FCS 5 ml
1%PS 0.5ml

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