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Biocompatibility Test

The document outlines the MTT assay procedure used to assess the cytotoxicity of prosthetics by measuring cell viability and metabolic activity. Key steps include cell seeding, treatment application, MTT reagent addition, solubilization of formazan, and absorbance measurement. Initial observations indicate that Sample 3 shows the highest cell viability after 24 hours, but further data is needed after 3 and 7 days for conclusive results on cytocompatibility.

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Amarjeet Kumar
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0 views

Biocompatibility Test

The document outlines the MTT assay procedure used to assess the cytotoxicity of prosthetics by measuring cell viability and metabolic activity. Key steps include cell seeding, treatment application, MTT reagent addition, solubilization of formazan, and absorbance measurement. Initial observations indicate that Sample 3 shows the highest cell viability after 24 hours, but further data is needed after 3 and 7 days for conclusive results on cytocompatibility.

Uploaded by

Amarjeet Kumar
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as KEY, PDF, TXT or read online on Scribd
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Biocompatibility Test

(MTT Assay)
For cytotoxicity of prosthetics.

Amarjeet; 8th May 2025


Procedure & Key Steps
The MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
assay) is a colorimetric assay commonly used to assess cell metabolic
activity as an indirect measure of cell viability, proliferation, or cytotoxicity.
It is based on the reduction of the yellow tetrazolium salt (MTT) to purple
formazan crystals by mitochondrial succinate dehydrogenase enzymes in live
cells.
Materials Used
• Cultured cells (L929, HeLa) to be confirmed)
• 50-well plate
• MTT reagent (5 mg/mL in PBS or culture medium)
• Culture medium (DMEM)
• Solubilization agent (DMSO)
• Microplate reader (570 nm)
Step by Step Procedure

1. Cell Seeding
Seed cells (typically 5,000–10,000 cells per well) into a 50-well plate.
Incubate for 24 hours to allow cells to adhere and recover.
1.

2. Treatment Application
The media was replaced with our test material (PDMS for cytotoxicity testing).

It included control wells:


• Negative control (untreated cells)
• Positive control (known cytotoxic agent)
Incubated for 24 hours so far. However the process will continue for 7 days.
3. MTT Reagent Addition
• 20 µL of MTT solution was added to each well.
• Incubated for 3–4 hours at 37°C in a CO₂ incubator.
• Viable cells reduce MTT to purple formazan.

4. Solubilization of Formazan
• Carefully the medium was removed.
• Addition of 100 µL of DMSO to dissolve formazan crystals.
• The plate was gently shaken for 5–10 minutes to ensure complete dissolution.
5. Absorbance Measurement
• The absorbance was measured at 570 nm using a microplate reader.
• A reference wavelength was used for background subtraction. (630-690 nm)

Data Interpretation
• The intensity of the purple color is directly proportional to the number of
viable cells.
• % Cell Viability = (OD of test sample / OD of control) × 100
• A lower absorbance indicates higher cytotoxicity.
Data collected after 24 hours.
Observations
After 24 hours the Sample 3
has shown highest cell viability
and hence highest metabolic
activity.
However it is still in early stage
and we need two more data
(1st after 3days and 2nd after
7days) to provide concluding
remark on cyto-compatibility of
our prosthetics.

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