Cell Culture Bewo Trophoblasts Biology Essay

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Hams-F12 growth media containing L-glutamine, with addition of FBS was used to grow and maintain Bewo trophoblasts. Anti-biotics; Penicillin and Streptomycin were also added to growth media to avoid bacterial contamination.

Jeg-3 Trophoblasts

EMEM growth media with addition of FBS, was used to grow and maintain Jeg-3 trophoblasts.

Penicllin & Streptomycin were also added to media to avoid bacterial contamination, along with Non-essential amino acids, Sodium pyruvate and L-glutamine.

Cell culture growth and maintenance

Both trophoblastic cell lines used the same cell culture procedure's described below with corresponding growth medias.

Approximately 200,000 cells were placed in a t25 cell culture flask containing 10mls of growth media. Cells were left to grow for a period of 2-4 days depending on confluence of cells. Upon full confluence, cells were washed ~ 3 times, using PBS (~2-3ml) to remove any media still present in flask, enabling easy detachment of cells. Once cells were washed, 2-3 ml of Trypsin (x2 dilution) was introduced to detach cells by incubation, for ~ 5 minutes at 37°C. Cells were viewed under the microscope to check cell detachment, followed by brisk addition of 2-3ml of growth media to stop action of trypsin exerting cell damage via protease activity. Once this was completed, cells were extracted via pipetting and placed into a sterile 15ml plastic tube, and centrifuged at 12000rpm for 5 minutes, for collection of cell pellet. Upon completion of centrifugation, cell pellet was re-suspended in 1ml growth media, to achieve single cells. This was carried out in order to attempt cell counting using a haemocytometer.

Cell counting by haemocytometer methods

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After re-suspension of cell pellet, a 1 in 10 dilution of original cell suspension was prepared to carry out cell counting. This dilution method was manageable for cell counting, as large cell pellets contain millions of cells difficult to count without dilution. The serial dilution was prepared as follows:

20µl original cell suspension + 180µl Corresponding growth media

Cell dilution was briefly vortexed for even distribution of cells. From this 20µl of cell dilution were pipetted onto the haemocytometer grid. Cells were counted using a cell counter. Figure 3a represents a haemocytometer grid used for cell counting.

Figure 3.3.1: Representation of Haemocytometer grid.

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Figure 3.1 shows a representation of a haemocytometer grid, areas shaded in light red show the areas in which cell were counted in, a total of 5 areas were used. The following formula shows how a total cell number was calculated.

Formula to obtain total cell count;

Average of all green grids x dilution factor x 104 (Constant) = No. of cells per ml

To obtain cell/µl = No. of cells per ml

1000

To calculate volume of cell suspension needed to seed 200,000 cells into cell culture flask; 200,000

Cells per/µl

Calculated cell suspension volume of original cell suspension was placed into t25 flask containing 10ml growth media, and placed in cell culture incubator at 37°C. This was used to maintain cells and obtain cells for seeding into 96 well plate in preparation for MTT analysis.

BPA Treatments

Corresponding BPA treatments administered to both trophoblasts were identical, the vehicle used in this experiment was 0.05% methanol. Treatments and 96 well plate layout are represented in Table 3.2 and Figure 3.3 respectively.

Table 3.3.2 BPA Treatments

Treatments

Prepare growth media (containing vehicle 0.05% methanol) = 25ul methanol in 50ml Growth media

BPA alone

 

Stock BPA (ug/ml)

Stock BPA (ng/ml)

Volume of BPA (ul)

Growth media (ul)

Serum free media (ul)

Total (ul)

Final BPA conc (ng/ml)

0

0

0

0

1000

1000

20

2

2000

10

990

1000

100

2

2000

50

950

1000

200

20

20000

10

990

1000

2000

20

20000

100

900

1000

10000

1000

1000000

10

990

1000

Prepare serum free media (containing vehicle 0.05% methanol)

= 25ul methanol in 50ml Serum free media

Serum Free + BPA

 

Stock BPA (ug/ml)

Stock BPA (ng/ml)

Volume of BPA (ul)

Growth media (ul)

Serum free media (ul)

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Total (ul)

Final BPA conc (ng/ml)

0

0

0

0

1000

1000

20

2

2000

10

990

1000

100

2

2000

50

950

1000

200

20

20000

10

990

1000

2000

20

20000

100

900

1000

10000

1000

1000000

10

990

1000

Figure 3.3.3 Plate Layout for BPA Treatments

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Figure 3.3.3 outlines specific BPA doses administered in the 96 well plate in a columnar fashion. Untreated control cells grown in the far left hand side column followed by series of different BPA doses dependant treatments.

NB: Growth media described as Serum free media for BPA treatments did not contain FBS. Hams-F12 growth media for Bewo cells, contained Penicillin and Streptomycin only.

EMEM growth media contained the following; Penicillin & Streptomycin, Non-essential amino acids, Sodium pyruvate and L-glutamine only.

Seeding cells into 96 well plate

Bewo and Jeg-3 trohpoblasts were both prepared and counted as described in the previous procedure, for the preparation of BPA treatments and MTT analysis.

Depending on the day of plating different cell densities were placed into the 96 well plate. Table 3.4 and 3.5 outlines the layout of preparing trophoblasts for MTT Analysis.

Depending on the day of plating cells were plated into 96 well plates, for MTT analysis accordingly shown in the tables below;

Table 3.3.4 : Plating of Bewo Trophoblasts for 72 hours BPA Exposure

Day of plating 1st Treatment 2nd Treatment 3rd Treatment MTT Analysis

Number of cells

Seeded per well

2,000 Friday Following Monday Tuesday Wednesday Thursday

Notes

Cells were left to grow in tissue culture incubator at 37oC on Saturday and Sunday.

Each treatment was left for 24 hour exposure.

Each treatment was left for 24 hour exposure.

Each treatment was left for 24 hour exposure.

MTT analysis was carried out and analysed on the fourth day after execution of all three treatment doses.

5,000 Monday Tuesday Wednesday Thursday Friday

Notes

If cells could not be plated on Friday, a higher cell density was plated out on Monday, left overnight for cells to grow.

Each treatment was left for 24 hour exposure.

Each treatment was left for 24 hour exposure.

Each treatment was left for 24 hour exposure.

MTT analysis was carried out and analysed, after execution of all treatments.

3.4 MTT Analysis

The 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay is a colorimetric assay, measuring the activity of mitochondrial enzymes reducing MTT to formazan dyes, to produces a purple precipitate. This procedure enables the assessment of viability and proliferation of cells, and can also determine cytotoxicity of toxic agents, identifying whether they are able to induce or inhibit cell viability and growth, (Abate et al., 1998).

Aims of MTT

The objectives of MTT in this particular research were to establish cell viability of both trophoblasts, (Bewo & Jeg-3) exposed to BPA Alone and in serum starvation with BPA at different concentrations. MTT absorbance values measured t 570nm, indicates the status of cell viability.

Upon completion of BPA treatments, 10µl of MTT was added to each well (of the 96 well plate), and incubated for 60 minutes in cell culture incubator at 37°C. After incubation, the remaining media was extracted via pipetting, and 100µl of DMSO was added to dissolve the MTT precipitate. The cell culture plate was then placed in the micro plate reader and absorbance's were measured at 570nm. NB No MTT was added to the wells indicated as Blank, but the media was removed and 100µl of DMSO was added to measure absorbance's.

Data collated was then analysed, by misusing the average blank value from all wells, this was done to eliminate and absorbance interfering with values due to the plastic ware of the micro plate. Percentage viability was calculated against percentage viability of untreated control cells.

Reduction of tetrazolium salts is now broadly accepted as a reliable procedure to examine cell viability. The MTT colorimetric assay is a rapid and time efficient technique, which requires no extra washing steps, and can be completed using one micro plate, lessening the need of additional plastic ware. Furthermore the absorbance's of each cell treatment is measured by a plate reader and the micro plate is discarded upon completion. This colormetric technique relies on several factors such as; enzyme activity, ATP production, co-enzyme production along with nucleotide uptake. A higher cell viability percentage suggests cells possess high metabolic activity, as mitochondria are able to generate ATP, in contrast a low percentage of cell viability suggests decreased amount of ATP production with decreased metabolic activity, indicating the toxin in investigation is influencing cell processes, (Abate et al., 1998 , Mueller et al., 2004.

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Metabolic activity of proliferating cells is higher proliferating cells in comparison to non-proliferating cells, therefore the MTT not only determines, cell viability, factor induced cytotoxicity but also identifies cell activation and proliferation. Figure 3.7 Actions of MTT within a viable (active) cell:

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Figure 3.7 Show the addition of MTT molecules (yellow) received by mitochondrial dehydrogenase enzymes, converted to MTT formazan crystals (Purple), indicating the cell is viable and able to produce ATP (orange).

Reduction of MTT only takes place when mitochondrial dehydrogenases enzymes are active. Fluctuations in metabolic status of cells after treatment with an agent can generate large changes in MTT. The yellow MTT tetrazole, in living cells is reduced to purple formazan in living cells by active mitochondrial dehydrogenases. MTT is cleaved to produce formazan via the metabolic enzyme; succinate-tetrazolium reductase, this enzyme belongs to the mitochondrial respiratory system and is only present in active viable cells. DMSO is administered after cell incubation with MTT to dissolve the insoluble purple formazan product, producing a purple coloured solution. Absorption of dissolved formazan in this visible region correlates with the percentage of intact living cells. The comparison between the purple formazan formed in treated cells and untreated cells, can be used to assess viability and changes in cell metabolism. Toxins are able to damage and harm cells which will lessen the reduction of MTT to formazan. ATP plays a vital role in energy transfer, and is the main reserve for cells energy to undertake many cellular reactions such as; transport, biosynthesis and movement processes, (Abate et al., 1998 and Mueller et al., 2004).

3.5 Protein estimation & SDS-PAGE

The aim of protein estimation by Lowry's method was to determine the concentration of the proteins in question. This will thus dictate the volume of the cells samples containing equal amounts of protein required to be loaded into the SDS-PAGE gel, to complete the last stage of the study; Western Blotting.

To determine the concentration of protein present in each sample the Lowry's method was implemented. The explanation behind this procedure is that the phenolic group belonging to the tyrosine and tryptophan residues present in the protein, will produce a blue-violet coloured complex holding maximum absorption at 750nm, containing Folin-Ciocalteau reagent comprising of sodium tungstate molybdate and phosphate. The colour intensity produced is dependent on the quantity of aromatic amino acids present and vary in different protein samples, (Wilson, and Walker, 2000).

Initiation of protein estimation was carried out by seeding ~200,000 cell in four t25 cell culture flasks.

The order of t25 flasks are outlines below in table 3.7

Table 3.8 Preparation of Cell culture for Protein estimation, SDS-PAGE and Western Blotting

Flask 1: Untreated control cells

Flask 2: Bewo/Jeg-3 trophoblasts with 200ng/mL

Flask 3: Serum starved Bewo/Jeg-3 Trophoblasts

Flask 4: Serum starved Bewo/Jeg-3 Trophoblasts with 200ng/mlL

Table 3.8 Same procedure was used for Bewo and Jeg-3 cells. Bewo cells were grown in the above conditions for a total of 72 hours and Jeg-3 cells were grown for total of 96 hours. Upon completion of exposure period, cells were scraped off and cell pellet was obtained. Cell pellet was centrifuged at 12,000 rpm for 5 minutes and re-suspended in 1ml PBS, followed by centrifugation at 2,000rpm for 5 minutes. After centrifugation the cell pellet was washed with 1ml PBS (not re-suspended) and was centrifuged at 2,000rpm for 5 minutes. All PBS was removed from cell pellet and cell pellet was left to dry. Cell pellets were frozen until protein estimation apparatus was available to carry out protein estimation, SDS-PAGE and Western Blotting.

After protein estimation apparatus was assembled, all cell pellets were collated, and upon cell pellet 100µl-150 µl total lysis buffer was, (smaller pellets required less amount of total lysis buffer and vice versa) added along with 20 µl protein inhibitor. Samples were then heated at 95°C for 5 minutes, and corresponding amounts of sample containing equal amounts of protein were added to separate 1.5 ml eppendorf tubes, the same amount of loading dye was added to the samples.

In this investigation Bovine Serum Albumin (BSA) was used to prepare the standard curve for proteins. BSA is a universally accepted standard protein, low in cost, containing high purity and conveniently available. This method is approximately sensitive to around 10µl/ml and is broadly used in various laboratories. This reaction is pH dependent, critical for its mechanism of action, the pH required is between 9 to10.5, (Wilson and Walker, 2000).

Firstly the standard curve was prepared. The working Lowry reagent was prepared by making 100ml of sodium carbonate/sodium hydroxide solution, this was carried out by adding 2.0g of sodium carbonate and 0.4g of sodium hydroxide to 100ml distilled water. Next reagent to be prepared was the Folin Ciocalteau reagent which was based on a 1:1 ratio of Folin Ciocalteau reagent to distilled water, (i.e. 1ml Folin Ciocalteau reagent added to 1ml distilled water). The working Lowry reagent was added to all protein samples including Bewo and Jeg-3 samples and inverted to mix, this was incubated for a duration of 15 minutes.

After incubation 0.1 ml of diluted Folin-Ciocalteau reagent was added to all samples and vortex mixed. Samples were then left for a minimum of 30 minutes at room temperature.

After samples had been incubated for a minimum of 30 minutes, 100µl of each sample was placed into a micro plate in duplicate and absorbance's were measured at 750nm. From this data a calibration graph of the standard proteins was prepared, which enabled the protein concentration of the unknown samples (Bewo and Jeg-3) to be calculated.

As per the protein estimation calculations, 20µl of protein of each sample was calculated and loaded into the SDS-gel wells.

Each well in the SDS-PAGE gel could accommodate up to 45 micro liters, cell samples loaded had to be within this limit. A 12% SDS gel was used in all SDS-PAG electrophoresis's carried out. From this western blotting was able to be implemented.

3.6 Western Blotting

Upon completion of SDS-PAGE, the SDS gel was transferred to a nitrocellulose membrane, the method of transfer was electro-elution. For every 100mm2, a current of 100mili amps was exerted. The electric current varied as in the first western blot attempt, two SDS-gels were used whereas in the second attempt four SDS-gels were used. Therefore a current of 200 mile amps and 400 mili amps was used respectively.

Electro elution uses mobility of proteins to imprint them onto the nitrocellulose membrane, including direct of the gel and membrane, (Wilson, K. and Walker, J. (2000). The gel was stacked as follows: Four pieces of filter paper were placed underneath the nitrocellulose membrane followed by addition of the SDS-gel layered on top of the membrane, with additional four pieces of filter covering the SDS-gel, sandwiching all the components of the transfer together. The gels were left for 60 minutes for samples to be transferred onto nitrocellulose membrane. After the transfer period was complete, the polyacramide gel was disposed of in the incineration container.

Upon transfer completion the nitrocellulose membrane was placed in the primary Nrf2 antibody overnight and refrigerated. The following day, the primary anti-body was placed into a separate container and washed with PBS tween for series of 5 times on the plate shaker, for an interval of 10 minutes for each wash.

After washing, reversible copper stain was applied to the membrane to evaluate transfer of proteins. 4 to 5 drops of the copper phthalocyanine 3,4',4',,4''' tetrasulphonic acid terasodium salt - 0.05 % (w/v) in 12 mM HCl (copper stain) were placed on the membrane and agitated. The surplus copper stain was placed back into it original container and blot was washed with distilled water. The blot was then cut at 50kb using the protein ladder as a guide to separate the Nrf2 and GAPDH compartment, placed in the primary antibody accordingly. The blot

was then de-stained using 12mM NaOH until the copper stain had disappeared. The blot was rinsed with distilled water followed by TBS tween.

Immunoprobing of Western Blot and Blocking of Nonspecific Sites

The membrane used possesses a high affinity for proteins. Therefore it is vital to block the surfaces non-specific to the protein of interest. Gloves should be worn at all times whilst handling gels and nitrocellulose membranes, as skin contains many protein which can be a possible source of contamination, (Wilson, K. and Walker, J. (2000).

After the blot had been washed, 10ml of the blocking solution containing to secondary antibody; anti-rabbit antibody recognising Nrf2, dissolved in 3% milk marvel, was added to the blot to obstruct any non-specific sites, for a period of 2 hours at room temperature, placed on the plate shaker.

Upon completion of the 2 hour incubation, another series of 5 washes followed with TBS tween for 10 minutes each whilst placed on a plate shaker. The blot was then washed with distilled water for 2 minutes.

Chemiluminescence detection

The final stage of Western blotting consisted of chemiluminescence detection, this was carried out by addition of substrate solution, (33µl of Nbt and 44µl of BCIP), covering the whole blot, placed on the plate shaker. The blot was incubated for several minutes (~2-6 minutes). Detection of the control GPADH and protein in question were awaited. Finally the blot was removed and dried with paper towels and placed in a plastic sleeve for imaging and densitometry