Introduction MTT Assay

7/31/2019 Introduction MTT Assay

1/10

6.1 ANTIANGIOGENIC ACTIVITY

INTRODUCTION

The chicken embryo chorioallantoic membrane assay has been a model for studyingneovascularization since the early 1970s when it was adapted by Folkmann et al.

Assay Description

While there have been many variations developed over the years, the basic assay is performed by

implanting a membrane or coverslip containing the compound of interest on the chick embryochorioallantoic membrane through a hole cut in the egg shell. The subsequent incubation period

ranges from 1-3 days, depending on the compound, after which time angiogenesis can be

quantified via image analysis or colorimetric detection methods.

The CAM assay is quick, technically simple, and inexpensive. However, a major drawback is

that it is labor intensive due to the large number of eggs that are required to obtain consistent

results. The investigator should note that this is a non-mammalian system which should be taken

into consideration when interpreting results.

5.3.1 Test protocol

5.3.1.1 Antiangiogenesis study by chorioallantoin membrane (CAM) assay

CAM assay is routinely used as a preliminary method to determine antiangiogenic

effect of a compound (S. Taylor 1982, R. Crum 1985, M.A. Moses 1990). This assay is basedupon the formation of a chorioallantoic membrane, in which neovascularization takes place, in

fertilized chicken eggs at a certain stage of the development of the embryo. Agarose pellets

impregnated with the test compound are placed onto the vascular membrane of opened eggs, and

the influence on angiogenesis is evaluated (D. H. Paper 1999). For assay purpose the fertile

chicken eggs were procured from Kalchina hatchery, Modinagar.

5.3.1.1.1 Requirements

Fertile Chicken Eggs Agarose 70% EtOH -1,4-galactan sulfate


2/10

5.3.1.1.2 Procedure

Twelve eggs were used per experiment to test one compound as a given dose. The

eggs were fertilized at 370C and 80% relative humidity in ideal conditions. The shells of eggs

were cleaned with 70% EtOH to avoid infections. After 72 hrs 8-10 ml of albumin was removedwith a syringe at the lower side of the egg, and the hole was sealed with tape. Subsequently the

upper part of the shell was removed, and the eggs were covered with a plastic film and

incubated for another 72 hrs. At this point of time, when the diameter of CAM is between 1.8

and 2.6 cm, the pellets containing the test substances were placed on the CAM. Test substances

were dissolved or suspended in a 2.5% agarose solution. After gel formation, the volume of

agarose gel corresponding to the dose of the test compound to be applied to the CAM was

taken by means of a micropipette for viscous solutions. Therefore the agarose pellets do not

have a uniform size. The half-cone-shaped agarose pellets are fixed because they slightly sink

into the CAM. After 24 hrs the antiangiogenic effect was measured, either, after addition of

cream as a contrast fluid, by means of a stereomicroscope, and observing the avascular zone

surrounding the pellet, or, by observing avascular zone surrounding the pellet (if clear) bynaked eye. Antiangiogenic activity is expressed as a score where 0 = no or weak effect, 1 =

medium effect, and 2 = strong effect (capillary free zone is at least twice as large as the pellet).

Also membrane irritation and embryotoxicity can be evaluated. -1,4-galactan sulfate (LuPS S5)

with an average molecular weight of 20000 was used as positive control (R. Hoffman 1996) and

an agarose pellet as a blank.

5.3.2 Results and discussion

Effect of the test compounds on angiogenesis is recorded in Table 5.1. All the

compounds were tested at a dose of 10 g/pellet, i.e. having less than 40 nmol/pellet, because

at higher dose most of compounds showed a toxic effect. In (Z)-1-phenyl-2-(4-nitrophenyl)ethene analogues, compound 3a, 3d, 5a, 5b, 5c and 5d showed an antiangiogenic score of more

than 1. Compound 5a was found to be most potent with a score of 1.6 0.1 which is more than

that of standard. Compound 3a also showed good score, equal to that of standard. In (Z)-1-

(furan-2-yl)-2-(4-nitrophenyl) ethene analogues compounds 10 and 11h showed an

antiangiogenic score of more than 1. All other compounds showed antiangiogenic score less

than 1. Compound 11h was found to be most potent with a score of 1.3 0.1 which is

comparable to that of standard. No compound showed score greater than standard.

The result shows that synthesized compounds have significant antiangiogenic activity.

The most active analogues 3a, 3d, 5a, 5b, 5c, 5d and 10 have smaller groups like COOH,

COOCH3 or COCl as bridge substituents while the least active analogues 6e, 6f, 6g, 6i, 6j, 6m,

11c, 11e, 11g and 11k have comparatively large groups as bridge substituents (Potent activity

of compound 11h is exception in this regard). Compounds having aromatic substituents (6b, 6d,

6i, 6j, 6m, 11c-g), were least active. So, we can say that compound possessing the Piperidin-1-

ylcarbonyl, carboxyl, methoxycarbonyl and chlorocarbonyl moiety on (Z)-1-phenyl-2-(4-

nitrophenyl) ethene and (Z)-1-(furan-2-yl)-2-(4-nitrophenyl) ethene skeleton are among the


3/10

most active compounds in our study. We can say that size of bridge substituents, in the series

studied, affect the antiangiogenic activity.

In 4,5-disubstituted oxazole derivatives, Compound 15b, 15c, 15d, 15e, 15i and 15m

showed potent activity with an antiangiogenic score of more than 1. Other compounds were

not as much active. Compound 15d was most active with a score of 1.5 0.1, equal to that ofstandard. Results show that compounds, having 3,4,5-trimethoxyphenyl group on either

position (4 or 5) on oxazole ring, have potent activity. Results further supported already

reported data that the three methoxy groups are involved in binding of the molecule with the

active site. However, 4,5-bis(3,4,5-trimethoxyphenyl) analog (15a) was not as much active

which shows that the part of active site, at which second aromatic ring binds, does not have

same amount of affinity for the three methoxy groups, as they have for other one.

In 3-aroylindole analogues, compounds 21d and 21g were found to be most active,

and showed an antiangiogenic score of more than 1. All other compounds showedantiangiogenic score less than or equal to 1. In 1-aroylindole analogues, compound 23d, 23e

and 23f showed an antiangiogenic score of more than 1. All other compounds showed

antiangiogenic score less than or equal to 1. In this series compound 23d was found to be most

active with an antiangiogenic score of 1.8 0.1. Activity of compounds 21d, 21g, 23e and 23f

was also found to be potent. Results show that 1-aroylindole analogues are more active than

their corresponding 3-aroylindole analogues. This may be due to, either presence of

dimethylaminomethyl group at 3-position or 1-aroylindole configuration better suited for

binding to active site. Furthermore, compounds containing methyl group at 2-position were

more active than their corresponding unsubstituted analogues. It reveals that 2-position methyl

group plays an important role in binding of the molecule to the active site. In 3-aroylindole

analogues, compound containing nitro group at 5-position (21d) was more active thancorresponding chloro and bromo group containing analogues (21e and 21f). Good activity of

compound 21g showed that triisopropylsilyl group at 1-position increases activity i.e. a bulky

group at 1-position can also enhance/retain the activity. In 1-aroylindole analogues, all three 2-

methyl analogues (23d, 23e and 23f) showed potent activity. 5-Nitro analogue (23d) was most

active having a score greater than that of standard.

Table 5.1 Antiangiogenic activity of compounds in the CAM assay

Test compound Concentration Antiangiogenic scoreb

sd

(n = no. of experiment)(g/pellet) nmol/pellet

3a 10 28 1.4 0.1 (n =3)

4a 10 27 1.0 0.1 (n =2)


4/10

5a 10 26 1.6 0.1 (n =3)

6a 10 24 0.9 0.1 (n =2)

6b 10 22 0.4 0.2 (n =2)

6c 10 26 0.7 0.1 (n =2)

6d 10 22 0.3 0.3 (n =2)6e 10 21 0.6 0.1 (n =2)

3b 10 32 1.0 0.1 (n =2)

4b 10 31 0.8 0.1 (n =2)

5b 10 30 1.2 0.1 (n =2)

6f 10 29 0.6 0.1 (n =2)

3c 10 32 0.8 0.1 (n =2)

4c 10 30 0.6 0.2 (n =2)

5c 10 30 1.1 0.1 (n =2)

6g 10 29 0.3 0.4 (n =2)

3d 10 33 1.2 0.1 (n =3)

4d 10 31 0.9 0.1 (n =2)

5d 10 31 1.5 0.1 (n =3)

6h 10 28 0.8 0.1 (n =2)

6i 10 26 0.3 0.2 (n =2)

6j 10 23 0.2 0.3 (n =2)

6k 10 27 0.6 0.1 (n =2)

6l 10 27 0.5 0.1 (n =2)

6m 10 24 0.3 0.1 (n =2)

6n 10 20 0.6 0.1 (n =2)8 10 39 0.8 0.1 (n =3)

9 10 37 0.9 0.1 (n =2)

10 10 36 1.1 0.1 (n =3)

11a 10 37 0.8 0.1 (n =2)

11b 10 32 0.6 0.2 (n =2)

11c 10 28 0.2 0.3 (n =2)

11d 10 29 0.5 0.1 (n =2)

11e 10 27 0.3 0.1 (n =2)

11f 10 30 0.4 0.1 (n =2)11g 10 26 0.2 0.1 (n =2)

11h 10 31 1.3 0.1 (n =2)

11i 10 31 0.9 0.1 (n =2)

11j 10 27 0.6 0.1 (n =2)

11k 10 23 0.2 0.1 (n =2)

15a 10 25 0.8 0.1 (n =3)


5/10

15b 10 28 1.2 0.1 (n =2)

15c 10 29 1.3 0.1 (n =2)

15d 10 26 1.5 0.1 (n =3)

15e 10 28 1.2 0.1 (n =2)

15f 10 33 0.4 0.2 (n =2)15g 10 33 0.4 0.2 (n =2)

15h 10 29 0.6 0.1 (n =2)

15i 10 29 1.2 0.1 (n =2)

15j 10 33 0.4 0.2 (n =2)

15k 10 33 0.3 0.3 (n =2)

15l 10 30 0.7 0.1 (n =2)

15m 10 26 1.4 0.1 (n =2)

15n 10 29 0.5 0.1 (n =2)

15o 10 30 0.5 0.1 (n =2)

15p 10 26 0.6 0.1 (n =2)

21a 10 27 0.8 0.1 (n =2)

21b 10 25 0.8 0.1 (n =2)

21c 10 28 0.5 0.2 (n =2)

21d 10 26 1.1 0.1 (n =2)

21e 10 24 1.0 0.1 (n =2)

21f 10 27 0.8 0.1 (n =2)

21g 10 17 1.3 0.1 (n =3)

23a 10 24 1.0 0.1 (n =2)

23b 10 22 0.9 0.1 (n =2)23c 10 25 0.6 0.1 (n =2)

23d 10 23 1.8 0.1 (n =3)

23e 10 22 1.6 0.1 (n =2)

23f 10 24 1.3 0.1 (n =2)

Agarose pellet 0.1 0.1 (n =10)

-1,4-galactansulphate (LuPS

S5)

50 2.5 1.4 0.1 (n =10)

b0 = no or weak effect, 1 = medium effect, 2 = strong effect


6/10

Figure 5.1 A 7 day old chicken egg (Blood vessels are shown through a dissecting

microscope).


7/10

Figure 6.2 Figure showing capillary free zone for compound23d. Inner circle

representing approximate size of sample disc, and outer circle representing

approximate capillary free zone.

Introduction


8/10

MTT Cell Proliferation Assay Kit provides a simple method for determination of

cell number using standard micro-plate absorbance readers. Determination of cell growth

rates is widely used in the testing of drug action, cytotoxic agents and screening other

biologically active compounds. Several methods can be used for such determinations, but

indirect approaches using fluorescent or chromogenic indicators provide the most rapid

and large scale assays. Among such procedures, the MTT assay developed by Mossman

1983 is still among one of the most versatile and popular assays. The MTT assay involves

the conversion of the water soluble MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide) to an insoluble formazan. (Liu et al. 1997, Berridge et al.

1993, Vistica et al. 1991) The formazan is then solubilized, and the concentration

determined by optical density at 570 nm. The result is a sensitive assay with excellent

linearity up to approximately 106 cells per well.

MTT Cell Proliferation Assay Kit provides enough material to perform 1000

individual tests using standard 96-well microplates. Following the protocol described

below, a complete assay requires an overnight incubation. However, with a slight

modification, the whole procedure can be performed in five hours (not including cell

preparation time). For additional information concerning the numerous variations and

modifications of the MTT assay, we recommend that you consult the citations provided.

(Garn et al. 1994, Carmichael et al. 1987, Twentyman et al. 1987, Tada et al. 1986)

Materials

Kit Contents


9/10

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MW = 414, Component A),10 vials, each containing 5 mg

SDS sodium dodecyl sulfate (MW = 288, Component B), 10 vials, each containing 1 gmStorage and Handling

Upon receipt the kit should be stored at 4C protected from light. Stored properly, the kit

components should remain stable for 12 months.

Materials Required but Not Provided

Phosphate-buffered saline (PBS), sterile HCl, 0.01 M solution dimethylsulfoxide (DMSO)optional

Experimental ProtocolReagent Preparation

1. Prepare a 12 mM MTT stock solution by adding 1 mL of sterile PBS to one 5 mg vial of MTT(Component A). Mix by vortexing or sonication until dissolved. Occasionally there may be some

particulate material that will not dissolve; this can be removed by filtration or

centrifugation. Each 5 mg vial of MTT provides sufficient reagent for 100 tests, using 10 L ofthe stock solution per well. Once prepared, the MTT solution can be stored for four weeks at 4C

protected from light.

2. Add 10 mL of 0.01 M HCl to one tube containing 1 gm of SDS (Component B). Mix thesolution gently by inversion or sonication until the SDS dissolves. Once prepared, the solution

should be used promptly. Each tube makes sufficient solution for 100 tests, using 100 L perwell.

Culturing Cells

The culture conditions used to grow the cells can affect the results and must be taken into

consideration when analyzing the data. The age of the cultures, number of passages and detailsof the growth medium can all be important factors. Natural variation in the requirements and

growth rates of different cell lines make it difficult to provide precise guidelines for preparing

your cells. In general, cells seeded at densities between 5000-10,000 cells per well should reach

optimal population densities within 48-72 hours. Note that the presence of phenol red in the

final assay samples can seriously affect results. We strongly recommend that the cells becultured in medium free of phenol red, if possible. Alternatively, the final incubation with the

MTT can be performed after exchanging the cells into medium free of phenol red.

Labeling Cells


10/10

1. For adherent cells, remove the medium and replace it with 100 L of fresh culture medium. Fornon-adherent cells, centrifuge the microplate, pellet the cells, carefully remove as much mediumas possible and replace it with 100 L of fresh medium.

2.

Add 10 L of the 12 mM MTT stock solution (prepared in step 1.1) to each well. Include anegative control of 10 L of the MTT stock solution added to 100 L of medium alone.

3. Incubate at 37C for 4 hours. At high cell densities (>100,000 cells per well) the incubation timecan be shortened to 2 hours.

4. Add 100 L of the SDS-HCl solution (prepared in step 1.2) to each well and mix thoroughlyusing the pipette.

5.

Incubate the microplate at 37C for 4hours in a humidified chamber. Longer incubations willdecrease the sensitivity of the assay. (Niks et al. 1990)

6. Mix each sample again using a pipette and read absorbance at 570 nm.

Rapid Protocol Option

To shorten the time of the assay it is possible to use DMSO (not provided) as a solubilizing agent

to dissolve the formazan.6

1. After labeling the cells with MTT, as described above, remove all but 25 L of medium from thewells. For non-adherent cells it may be necessary to first centrifuge the plates to sediment the

cells.

2. Add 50 L of DMSO to each well and mix thoroughly with the pipette.3. Incubate at 37C for 10 minutes.4. Mix each sample again and read absorbance at 540 nm not 570 nm, as above.

References

Introduction MTT Assay

Documents

Transcript of Introduction MTT Assay