OVER-NIGHT CULTURE PREPARATION

The different bacterial strains – DH5α and B. Subtilis were sub-cultured in LB broth.
The Saccharomyces Cerevisiae strain was sub-cultured in Nutrient Broth.
Then the cultures were incubated overnight at 37°C in a shaker at 150 rpm.

GROWTH KINETICS

When the cultures reached OD~ 0.7 to 0.9, they were sub-cultured in a 96 well plate (CAT No: 934396) in their respective media as follows:

The cultures were incubated at 37°C in a shaker at 10rpm.
Absorbance was measured for every 30 minutes interval till the stationary phase was reached.

PEPTIDE DILUTION

1mM stock solution of peptides was prepared and 4mg of native and mutant Latarcin (Ltc 2a-Wt and Ltc 2a-Mut) peptides were dissolved in 1.378ml of dH2O respectively.
Concentration of stock solution = 1mM peptide.
Working solutions of Ltc 2a-Wt and Ltc 2a-Mut peptides were prepared from the stock.
A two fold dilution series of both peptides, ranging from 550 µM to 1 µM, were prepared by pipetting 500µL of each 550µM peptide solution into the 500 µL sterile nuclease and protease free type 1 water in 1.5ml eppendorf tubes.
The peptides were mixed by repeated pipetting 5-8 times.
A final volume of 220µM was prepared in the 96 well plate and the 20µL of the working stocks were added respectively to get a Dilution of 50µM to 0.1µM Ltc 2a-Wt and Ltc 2a-Mut.

ANTIMICROBIAL ACTIVITY

MINIMUM INIBITORY CONCENTRATION [MIC-ASSAY]

The overnight culture (OD ~ 0.7 to 0.9) of bacterial strains – E. coli DH5α and B. Subtilis and Saccharomyces cerevisiae were sub-cultured in LB broth and Nutrient broth respectively in a 96 well plate.
The cultures were incubated at 37°C at 10 rpm.
Absorbance were measured at 630nm for every 30 min.
After 6 hours (mid log phase), the peptides (both native and mutant) were added at different concentrations to their respective wells.
Absorbance measurement was carried out till the negative control (Media + Inoculum) reached the stationary phase.

HAEMOLYTIC ACTIVITY

HAEMOLYTIC ASSAY

The haemolytic activity of Latarcins-2a Wt and Mut peptide were determined using fresh bovine blood.
1ml of defibrinated blood was washed twice with 5 mL of 1X Phosphate Buffer Saline (PBS) and centrifuged at 4000 rpm for 10 min at 4°C and the supernatant was discarded.
A solution of 1% RBC was made by adding 100 mL of blood in 10 mL of PBS buffer.

PEPTIDE DILUTION AND ASSAY

In a sterile 96-well microtiter plate (U bottom plate), Ltc 2a-Wt and Ltc 2a-Mut peptides were prepared at concentrations 2X (128µM), by adding 25.6 µL of peptide stock to 74.4 µL of PBS in column 1.
A twofold dilution series of both peptides, ranging from 128µM to 0.065 µM, were prepared by pipetting 50 µL of each 2X peptide solution into the 50 µL PBS in column 2.
Using the pipette, the peptides were mixed by repeated pipetting 5-8 times.
50 µL was withdrawn from column 2 and added to column 3 which contains 50 µL PBS. Mixed using pipette and transferred to column 4.
The same procedure was repeated till column 12 and 50 µL from column 12 is discarded.
50 µL of 1% RBC was added to all wells till column 12, also to the three control wells which contain only PBS (control).
The final concentrations were in the range of 128 µM to 0.0625 µM from column 1 to 12 respectively.
The plate was incubated at 37°C and the readings were taken after 24 h.
Haemoglobin release was monitored by measuring the absorbance of the supernatant at 414 nm in the 96-well plate reader.

CYTOTOXIC ACTIVITY

MTT ASSAY

MTT assay was carried out in C2C12 (murine skeletal muscles) and HELA (cervical cancer) cells according to the protocols given below.

REVIVING THE CELLS

The cells were plated in 25-mm² T-flasks after thawing them at 37 °C in a water bath for few minutes.
The thawed cells were transferred to a centrifuge tube and centrifuged at 1200 rpm for 5min.
The cells were collected by discarding the supernatant and mixed well in Dulbecco’s Modified Eagle’s Medium (DMEM) solution with 10% fetal bovine serum, and antibiotics.
They were plated in 25 mm² T-flasks in the same DMEM solution, individually. The T-flasks containing the cells were incubated for 24 h in a humidified environment with 5% CO2.These cells were washed with phosphate buffer saline (PBS) after 24 h.
The medium was regularly changed to enable the cells reach 30-40% confluence.

CHECKING THE VIABILITY OF THE CELLS

The cells were seeded in a 96 well plate with 3000-4000 cells/well and incubated overnight in a CO2 incubator at 370C for 24 hr with 5% CO2. After incubation the cells were treated with Ltc 2a-Wt and Ltc 2a-Mut.
The cells were treated with different concentrations (4, 8,16,32,64,120 μM) of the Ltc 2a-Wt and Ltc 2a-Mut respectively.
The plates along with the treated cells were again incubated for 24 h and 48 h in the same conditions.
MTT solution was prepared (5mg/ml) and added to the control and treated cell line samples, and incubated again at 37 °C for 4 h. After incubation, the media was carefully removed from the wells and the cells were mixed with solubilizing buffer.
A purple colour solution was visible at this stage. The control solution and treated samples were set for shaking (~10min) in the plate reader to ensure that the formazan precipitate was dissolved. The measurement of MTT assay was analysed with a Plate Reader (Perkin Elmer) at 570 nm.
Percentage inhibition of cells was calculated as follows:

Cytotoxicity (%) = (experimental absorbance 570nm of exposed cells/absorbance 570nm of un-exposed cells) ×100

REFERENCES

Sergey A. Kozlov, Alexander A. Vassilevski, Alexei V. Feofanov, Andrey Y. Surovoy, Dmitry V. Karpunin, and Eugene V., Grishin (2006), Latarcins: Antimicrobial and Cytolytic Peptides from the Venom of the Spider Lachesana tarabaevi (Zodariidae) Exemplify Biomolecular Diversity, JBC Papers.

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Anton A. Polyansky*,1, Alexander A. Vassilevski1, Pavel E. Volynsky, Olga V. Vorontsova, Olga V. Samsonova, Natalya S. Egorova, Nicolay A. Krylov, Alexei V. Feofanov, Alexander S. Arseniev, Eugene V. Grishin, Roman G. Efremov, (2009), N-Terminal Amphipathic Helix As A Trigger Of Hemolytic Activity In Antimicrobial Peptides: A Case Study In Latarcins, ELSEVIER FEBS Letters.

CYTOTOXIC ACTIVITY

CELL LINES AND CULTURE

The MTT assay was carried out in C2C12 (murine skeletal muscles) and HeLa (cervical cancer) cell lines.
The lyophilised cells were thawed at 37 °C in a water bath for few minutes.
The thawed cells were then centrifuged for 5min at 1200 rpm.
The cells were collected by discarding the supernatant and mixed well in Dulbecco’s Modified Eagle’s Medium (DMEM) solution supplemented with 10% fetal bovine serum (FBS) and antibiotics.
They were plated in 25 mm²T-flasks in the same DMEM solution, individually. The T-flasks containing the cells were incubated for 24 h in a humidified environment with 5% CO₂. The cells were passaged when >90% confluence was reached.

CHECKING THE VIABILITY OF THE CELLS

The cells were seeded in a 96 well plate having 3000-4000 cells/well and incubated overnight in a CO₂ incubator at 37 degrees for 24 h with 5% CO₂.
After incubation, the cells were treated with different concentrations (4,8,16,32,64,120 μM/ml) of BP-100- . The non-treated cells and the cell-free media were used as control and blank respectively.
The plates were incubated again at the same conditions for different time intervals 24 h and 48 h.
Freshly prepared MTT solution (5mg/ml) was added to both control and treated wells and incubated at 37 °C for 4h. After incubation, the media was carefully removed from the wells and mixed with solubilizing buffer.
A visible purple colour formation was observed. The plate was kept in the shaker (~10mins) in the plate reader to ensure that the formazan precipitate was dissolved. Then the absorbance was measured at 570nm using the Plate Reader (Perkin Elmer).
The percentage of cytotoxicity was calculated using the formula given below.

   Cytotoxicity (%) = (experimental absorbance 570nm of exposed cells/absorbance 570nm of un-exposed cells) ×100

GENE SILENCING

CELL LINES AND CULTURE

The human cervical carcinoma HeLa cell line was cultured in Dulbecco's Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 0.1 g/ml streptomycin.
The cells were kept at 37˚C in a humidified atmosphere containing 5% CO2.
The cells were passaged when >90% confluence was reached.

TRANSFECTION OF shRNA INTO THE HeLa CELL LINES

The cells were seeded in a 24 well plate prior to transfection and incubated overnight.
The shRNA was diluted as per the manufacturer’s instructions to prepare 100nM stock solution from which 10µM working solution was prepared.

LIPOSOME COMPLEX PREPARATION

In a clean tube, 30 μl of shRNA was diluted to 500 μl using 1X PBS.
In a separate tube, 30 μl of Lipofectamine 2000 was diluted with 470 μl of 1X PBS. The two solutions were mixed gently and incubated at room temperature for 5 min.
The shRNA and Lipofectamine 2000 were added at the ratio of 1:2 and mixed gently by pipetting and the mixture was incubated at room temperature for 30 min to form liposome complexes.
The liposome complexes were added into their respective plates carefully and mixed gently for 30 s.
The plates were kept in the humidified incubator at 5% CO
All the experiments were carried out in triplicate.

BP100-(KH)₉-shRNA MIXTURE

100 μl stock was prepared by dissolving 4mg of BP100- in 10.5 ml of Milli-Q water from which 10 μl concentration of the working solution was prepared.
2μl shRNA was added to increasing volumes of BP100- at various N/P ratios (0.1, 0.5, 1 and 2) and the solution was made up to 100 μl using autoclaved Milli-Q water.
The solutions were mixed thoroughly and allowed to stabilize for 30 min at 25 °C.
The Lipofectamine-shRNA and the BP100- -shRNA mix was added into their respective plates carefully and mixed gently for 30 s.
The plates were incubated in the humidified incubator at 5% CO
All the experiments were carried out in triplicate.

TOTAL RNA EXTRACTION

After 24 hours, the cells were trypsinized and harvested for gene expression analysis.
The cells were collected in MCF tubes and centrifuged at 1500 rpm for 5 min.
To the pellet, 500 μl of Dulbecco's phosphate-buffered saline (DPBS) buffer was added and mixed well by pipetting. They were given a spin at 9000 rpm for 5min.
The supernatant was removed and the cells were homogenized by adding 500 μl of Trizol reagent and completely dissolved.
Then the homogenized cells were incubated at RT for 5 min and 100 ml of chloroform/500 μL of Trizol was added and shaken vigorously for 15 sec.
The mixture was incubated at RT for 13 min and then centrifuged at 4°C, 13000 rpm for 15 min.
The aqueous phase was collected and transferred to new MCF tubes to which 250 ml of isopropanol reagent/500 μl of Trizol was added and vortexed for 10 seconds and then incubated at RT for 8 min.
The mixture was centrifuged at 13000 rpm for 9 min at 4⁰C – 25⁰ The supernatant was aspirated out and 500 ml of ethanol/500 μl of Trizol was added to the pellet.
The mixture was given a spin at 7500g for 10min and the pellet was air-dried at 37⁰C for 15 min.
To the pellet, 20 μl of RNase free water was added and incubated at 55-60⁰C for 5 min to completely dissolve the pellet.
The purity and quantity of RNA was measured using NanoDrop.

qRT PCR

cDNA synthesis was carried out by incubating 2.5 μg of RNA with 1 μl of oligo dT primers, 1 μl of dNTPs, 1 μl of reverse transcriptase in 4 μl of RNase transcription buffer, 2.5 μl of RNase inhibitor and the reaction was carried out according to the manufacturer’s protocol.
1 μl of cDNA was mixed with the Master Mix to perform gene expression analysis.
GAPDH was used as internal control for normalization.
Each PCR analysis was performed in sets of technical duplicates.
The analysis was carried out according to the manufacturer’s protocol.

REFRENCES

Yue-Li, W, Xin, S, and Di-Nan, H, (2017), Intron-specific mediated downregulation of surviving and promotion of apoptosis in HeLa cells, Oncology Letters
Chang, E., Qiang Zhu, M., and Dreze., R., (2007), Novel siRNA-based molecular beacons for dual imaging and therapy, J., 2, 424-425.
Islam, Md., Odahara, M., Yoshizumi, T., Oikawa, K., Kimura, M., Su’etsugu, M., and Numata, K., (2019), Cell-Penetrating Peptide-Mediated Transformation of Large Plasmid DNA into Escherichia coli, ACS synth. Biol.
Cell culture protocols, Hela and CHO cells, Woods Hole Physiology Course, 2006.

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Team:REC-CHENNAI/Protocol

OVER-NIGHT CULTURE PREPARATION

GROWTH KINETICS

PEPTIDE DILUTION

MINIMUM INIBITORY CONCENTRATION [MIC-ASSAY]

HAEMOLYTIC ASSAY

PEPTIDE DILUTION AND ASSAY

MTT ASSAY

REVIVING THE CELLS

CHECKING THE VIABILITY OF THE CELLS

REFERENCES

CELL LINES AND CULTURE

CHECKING THE VIABILITY OF THE CELLS

CELL LINES AND CULTURE

TRANSFECTION OF shRNA INTO THE HeLa CELL LINES

LIPOSOME COMPLEX PREPARATION

BP100-(KH)9-shRNA MIXTURE

TOTAL RNA EXTRACTION

qRT PCR

REFRENCES

BP100-(KH)₉-shRNA MIXTURE