L-6 rat myocyte cell toxicity Measured in Cell-Based System Using Plate Reader - 2138-06_Inhibitor_Dose_DryPowder_Activity
This is a counter screen to look for host cell toxicity for T. cruzi experiments conducted in L6 cells. ..more
Keywords: L-6 myocyte, toxicity, host cell
This is a counter screen to look for host cell toxicity for T. cruzi experiments conducted in L6 cells.
Expected Outcome: Compounds significantly suppressing Alamar blue, and thus are toxic to L-6 myocytes, are considered active.
In vitro cytotoxicity with L-6 cells. Assays were performed in 96-well microtiter plates, each well containing 100 ml of RPMI 1640 medium supplemented with 1% L-glutamine (200mM) and 10% fetal bovine serum, and 4000 L-6 cells (a primary cell line derived from rat skeletal myoblasts)( Page et al., 1993 and Ahmed et al., 1994). Serial drug dilutions of eleven 3-fold dilution steps covering a range from 100 to 0.002 ig/ml were prepared. After 70hours of incubation the plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. 10ml of Alamar Blue was then added to each well and the plates incubated for another 2 hours. Then the plates were read with a Spectramax Gemini XS microplate fluorometer (Molecular Devices Cooperation, Sunnyvale, CA, USA) using an excitation wave length of 536 nm and an emission wave length of 588 nm. The IC50 values were calculated by linear regression (Huber 1993) from the sigmoidal dose inhibition curves using SoftmaxPro software (Molecular Devices Cooperation, Sunnyvale, CA, USA). Podophyllotoxine is used as control.
C.Page C, M. Page and C. Noel. 1993. A new fluorimetric assay for cytotoxicity measurements in vitro.International Journal of Oncology 3: 473-476.
S.A. Ahmed, R.M. Gogal and J.E. Walsh. 1994. A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H] thymidine incorporation assay. The Journal of Immunological Methods 170: 211-224.
In vitro sensitivity assays: Cytotoxicity
Standard Cell Lines:
L-6 (rat skeletal myoblast cells)
Podophylotoxin (PPT); starting concentration: 0.1microg/ml
average IC50 = 0.006 microg/ml
Medium: RPMI 1640 + 10% FCS + 1.7microM L-Glutamine (850microl 200mM for 100ml)
Culture vessel: CostarTM 96-well microtiter plates
Incubation: 37 degrees C, 5% CO2
Compounds are dissolved in DMSO (SOP Nr. D1), unless otherwise specified by the supplier. The stock solution is 10 mg/ml and stored at -20 degrees C. Stocks are kept for 3 years. (Since DMSO is toxic, care has to be taken not to exceed a final concentration of 1% DMSO in the assay).
1. Add 100microl of medium to wells of columns 3, 6, 9 and 12 of a microtiter plate. These wells serve as controls.
2. 100 microl of a cell suspension of 4x104 cells/ml is added into the remaining columns (1 and 2, 4 and 5, 7 and 8, and 10 and 11). Cells are allowed to attach over night. Per plate, allow for 6.5ml of cell suspension to be used.
3. The next day, the medium is removed from row H (do this for half of the plates and go to step 4 and return to step 3 for the second half, so the cells don't dry out).
4. 150 microl of medium containing the highest drug concentration is added to the wells of row H. Four drugs can be tested on one plate (drug 1 column 1-3, drug 2 column 4-6, etc.).
5. Serial drug dilutions are prepared by using a 12-channel multi-pipette. First, remove 50 microl from wells of row H and put into row G and mix well. Next, 50 microl are taken out of row G and put into row F and so on until row B. The last 50 microl of row B are discarded. A serial dilution factor of 1:3 is thus obtained. Wells in row A serve as control wells without drugs.
6. The plates are then incubated for 70 hrs at 37 masculineC / 5% CO2.
1. The plates are inspected under an inverted microscope to ensure that growth is normal. Additional information may be recorded, such as drug insolubility or contamination, etc.
2. Add 10 microl of the fluorescent dye Resazurin to each well and incubate the plates for another 2 hours (until a color change is observed, but maximum 3 hours).
3. To determine an IC50 value, the plate is read at excitation wavelength 530 nm and emission wavelength 590 nm (pre-set LILIT template file). Make sure that the values in each well are approximately 10 times the background values.
Data are transferred into a graphic program (Excel) and are evaluated to determine the IC50 or analyzed using the fluorescent plate reader software (SoftMax).
Compounds with IC50 less than 100 fold greater than the control IC50 (Podophyllotoxin) were labeled active.
Categorized Comment - additional comments and annotations
* Activity Concentration.
Data Table (Concise)