Scribd
Upload
48 views4 pages

Cell Based Assay

Cellular assays, or cell-based assays, can be used in both biomedical research and drug-discovery screening applications to efficiently quantify cytotoxicity, biological activity, biochemical mechanisms and off-target interactions.

Uploaded by

rajapakshac252
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
48 views4 pages

Cell Based Assay

Cellular assays, or cell-based assays, can be used in both biomedical research and drug-discovery screening applications to efficiently quantify cytotoxicity, biological activity, biochemical mechanisms and off-target interactions.

Uploaded by

rajapakshac252
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
You are on page 1/ 4

PBMC isolation

• PBMCs were separated by centrifugation of blood diluted with RPMI-1640


medium over Histopaque (Ficol) 1077 (Sigma-Aldrich, St. Louis, MO) at
200g for 15 min.
• The interphase layer was collected by pipette and was washed in RPMI-
1640 medium.
• The pellet was resuspended to a final volume of 0.68 ml with RPMI-1640
medium.
• The cells were cultured within T25 culture flask for overnight in the
supplemented RPMI-1640 with fetal bovine serum (FBS), 100 U/mL
penicillin, 100 µg/mL streptomycin and 2 mM L-glutamine at 37°C for
24 hours before any treatments.
• Before performing the experiment, the medium was discarded; the
separated cells were washed and counted. The cell viability was measured
by trypan blue staining to greater than 95% viability.

Comet assay

• Comet assay under alkaline condition was employed to evaluate single and
double strand breaks at single cell level.
• In brief, 3.5 × 105 PBMC cells/well was treated with 2x IC50
concentrations of drug for 2 h.
• After collection and counting the cells, about 104 cells in a volume of
100 µl were gently pipetted in a 37 °C PBS solution containing 1% LMPA
(low melting point agarose), and were spread over a microscope slides
precoated with 1% conventional agarose under a coverslip, and chilled in
4 °C for 15 min to solidify.
• After removal of the coverslip, the slides were submerged for 1 h in cold
lysis buffer (10 mM Tris, 2.5 M NaCl, 100 mM EDTA and 1% freshly
added Triton X100, pH 10), and then immersed in denaturing buffer (1 mM
EDTA and 300 mM NaOH, pH 13) at 4 °C for 30 min to unwind the DNA
in the lysed cells.
• Subsequently, slides were electrophoresed under the same denaturing
condition for 30 min at 0.6 V/cm. after electrophoresis, slides were dipped
in neutralization solution (0.4 M Tris, pH 7.5) for 5 min, stained with
Ethidium Bromide(2 µg/ml) , and imaged by a fluorescent microscope
(Axioscope 2 plus, Zeiss, Germany).
• Open Comet software was used to analyze the Comet images, and the
presented data are the results of analysis of at least 100 comets in each
sample.

Trypan Blue assay

• Ten microliters (10 μl) of 0.4% Trypan Blue (w/v) and 10 μl of the
PBMCs suspension were transferred into a cryovials tube (dilution factor
= 2) and mixed,
• It was then allowed to stand for maximum of 5 min. While the cover-slip
was in place, the pipette was used to transfer 10 μl of Trypan blue-PBMCs
suspension mixture to both chambers of the haemocytometer.
• Both the viable and non-viable cells were counted using a light
microscope. Non-viable cells were stained blue, whereas viable ones
remain colourless. The percentage of viable cells was calculated.

MTT Assay

• The cytotoxic activity of compounds can be evaluated using the MTT


method described by Mosmann [18].
• PBMC (5 × 10 5cells/well) are cultured in 96-well plates in RPMI 1640
complete culture medium containing 10 % fetal calf serum (FCS) in the
presence of compound at different concentrations previously diluted in
culture medium, for 24 h at 37 °C, and under 5 % CO2.
• Next, 20 μL of MTT solution at 5 mg/mL isadded to each well, and the
plates are incubated for 4 h, at 37 °C, and under 5 % CO2.
• After incubation, the precipitated formazan crystals are dissolved with 200
μL of 20 % SDS (sodium dodecyl sulfate), and absorbance is recorded at
570 nm.

Haemolytic activity

• Collect blood in heparin or sodium citrate tubes [Sodium Citrate 3.2%]


tubes and immediately centrifuge at 1700× g for 5 min. Avoid using needles
above 23 G in order to minimize pre-analyte hemolysis.
• Remove the supernatant by aspiration and wash the erythrocytes by adding
2 mL of PBS pH~7. Centrifuge at 1700× g for 5 min. Repeat the washing
step three times or until supernatant is clear.
• Remove supernatant and dilute the erythrocyte pellet 1:100 in PBS pH~7
to obtain a 1% erythrocyte suspension.
• 250 μL of test compound, water, or detergent were mixed with 250 μL 1%
erythrocyte suspension in an Eppendorf tube.
• Samples were subsequently incubated at 37 °C for 60 min.
• After incubation, the tubes were centrifuged at 1700× g for 5 min, and then
50 μL of the supernatants were transferred to transparent, flat-bottom 96-
well plates
• absorption was measured at 405, 530, and 570 nm in a microplate reader.
• Triton X 100 10 % v/v van be used as the positive control
Anti-haemolysing effect (of antioxidants)

Peroxyl radical-induced haemolysis of erythrocytes

• AAPH–PBS solution (30 mM as the final concentration) was added to a


suspension of erythrocytes in PBS (3.0%, v/v) in which the DMSO solution
of antioxidant was added in advance to a certain concentration
• The suspension was incubated at 37oC. Samples were taken from the above
incubation mixture and centrifuged at 1700 g for 5 min to remove the
erythrocytes and obtain the supernatant.
• The percentage of haemolysis was determined by measuring the
absorbance of
• the supernatant at 540 nm (A540) and compared with that of complete
haemolysis in the absence of antioxidant
• To avoid the influence of DMSO on the haemolysis, it is worth noting that
the final volume of DMSO in all the experiments was 1.0% (v/v), and the
same amount of DMSO was added to the control experiment as well.

You might also like

pFad - Phonifier reborn

Pfad - The Proxy pFad of © 2024 Garber Painting. All rights reserved.

Note: This service is not intended for secure transactions such as banking, social media, email, or purchasing. Use at your own risk. We assume no liability whatsoever for broken pages.


Alternative Proxies:

Alternative Proxy

pFad Proxy

pFad v3 Proxy

pFad v4 Proxy