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  1. May 2019
    1. suspension through a 26 gauge needle. Lysates were cleared by centrifugation at 14,000 g for 30 min at 4°C and supernatant was used for protein estimation using BCA protein estimation kit (Pierce)
    2. P. Jalciparum infected erythrocytes were lysed by the addition of 0.05 % (w/v) saponin to release parasites, followed by a 30 minute incubation on ice. To remove debris and lysed RBCs were washed with cold PBS followed by centrifugation at 8000g. The lysis buffer containing 10 mM Tris pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100, and Ix complete protease inhibitor cocktail (Roche Applied Science) was added to the parasite pellet and homogenized by passing the
    3. Preparation of Parasite Cell Lysate
    1. at the acidic and basic endpoints of the titrations. Na+ free and HC03-free buffer were prepared as described by Khaled et al.
    2. Intracellular pH measurement was performed using the long-wavelength fluorescent pH indicator carboxy SNARF-1 AM. THP-1 macrophages were resuspended in serum-free and phenol-red free RPMI-1640 medium (106 cells/mL) and incubated at room temperature for 15 min with SNARF-1 AM at a final concentration of 1 f.!M. The cells were washed once in fresh serum-free media and incubated for 20 min for complete de-esterification of intracellular acetoxymethyl esters. In situ calibration ofSNARF-1 AM was performed to determine the pKa of the dye at 3 7°C by using the ionophore Nigericin (1 0 f.!M), which maintains the intracellular pH the same as that of the controlled extracellular medium in a buffer containing high-K+. Appropriate groups were subjected to different treatments and fluorescence measurements were commenced in a spectrofluorimeter (Perkin Elmer, Waltham, MA, USA) followed by kinetic analysis. The pH was calculated from the fluorescence measurements using the following formula: where pKa of carboxy SNARF-1 AM is 7.5 at 37 °C. R is the ratio of fluorescence intensities (F) measured at two emission wavelengths, 580 nm (AI) and 640 nm (A.2), with fixed excitation at 514 nm. The subscripts A and B represent the limiting values
    3. Measurement of intracellular pH
    1. The proteins were resolved on SDS-polyacrylamide gels and transferred onto a nitrocellulose membrane, in the transfer buffer, at a constant current of 300 rnA for 2 h. The membrane was incubated in blocking buffer for 45 min. at room temperature with continous shaking. The membrane was further incubated in anti-restrictocin antibody diluted in PBS, pH 7.4, containing 0.1% Tween 20 (PBST), for 45 min .. The membrane was washed thrice with PBST, followed by incubation in anti-rabbit IgG-HRP conjugate diluted in PBST for 30 min. with shaking. After repeated washings with PBS, colour was developed by incubating the membrane with the chromogenic substrate 0.5 mg/ml of DAB.4HCI (diaminobenzidine tetrahydrochloride dihydrate) and 1 f..ll/ml of H202 in PBS.
    2. Western Blotting
    1. eppendorf tube was put at the bottom of the column to collect the eluate. The column was respun as before and the purified probe collected in the eppendorf tube, the unincorporated nucleotides remaining within the column. One ul aliquot from the purified probe was diluted 100 fold, mixed well and 1 ul aliquots were put in triplicate into 3 ml scintillation fluid containing vials which were counted in a Beckman Liquid Scintillation Counter. The total radioactivity of the probe was calculated by multiplying the mean radioactivity of the three diluted samples with a factor of 104 ( dilution factor, 102, total reaction volume, 102 ). The specific activity of the probes ranged from 1 X 107 to 5 x·1o7 cpm 1 ug DNA. The probe purified by the above method did not require any further purification.
    2. The nick translated probe was purified by a spun column procedure to remove the unincorporated nucleotides. A sterile 1 ml syringe was plugged at the lower end with siliconised glass wool. The syringe was then filled with Bio-gel P-4 Bio Rad Laboratories, USA ) equilibrated in advance with TE. For doing this, 30 grammes of Bio-gel P-4 was slowly added into 250 ml of TE ensuring a good dispersion of the powder. This was then autoclaved at 15 psi for 20 minutes. After cooling, the supernate was decanted and replaced with an equal volume of sterile TE. The slurry was stored at 4°C. The slurry was poured upto the 1 ml mark in the syringe. The syringe was placed into a centrifuge tube and spun at 2000 · rpm for 3 minutes. The column was packed by repeating this process till the packed column volume reached 1 ml mark. Next, 50 ul of 2 mg 1 ml denatured salmon sperm DNA was loaded as carrier and the column spun as before. 100 ul of TE was then added to the column and it was respun as before. Finally, the nick translation reaction was diluted to 100 ul with TE and loaded on to the column. A sterile 1.5 ml
    3. Purification of the probe.
    4. 400 ci 1 mmole to 3000 ci 1 mmole. The nick translation reaction was set up as recommended by the manufacturer of the kit, using about 0.5 ug DNA. The reaction was incubated at 12 -14 °C for 90 minutes, except in the case of small fragments ( 500 bp ) when the reaction was incubated for 45 minutes only. The reaction was terminated by the addition of stop buffer supplied with the kit.
    5. DNA was labelled using the nick translation kits supplied by BRL or NEN, USA, or Amersham, UK. The 32P-deTP was from either NEN or Amersham, UK, at a concentration of 10 mei I ml. The specific activity of the label ranged from
    6. Nick translation.
    7. 32~ -labelling of DNA.
    1. TheO2carryingcapacity(Vol%)ofbloodwascalculatedby multiplyingtheHbcontentwith1.25O2combiningpowerofHb/g(Johansen,1970).
    2. TheO2carryingcapacity
    1. annealing at 25°C for 5 minutes, the reaction was incubated at 42°C for one hour.
    2. series of primers were designed to detect the levels of intact gene of interest or Rz in the cell lysate. The levels of RNA were quantitated by carrying out reverse transcriptase based-PCR using the Im.Prom-11™ Reverse Transcriptase system (Promega, U.S.A.). 1}lg of template RNA and 1}lM terminal primers were combined in 5pl reaction volume and the primer I template mix was thermally denatured at 70°C for 5 minutes and chilled on ice. A reverse transcription reaction mix of volume 15 pl was assembled on ice to contain nuclease-free water, 1X reaction buffer, 1pl reverse transcriptase, 6 mM magnesium chloride, 0.5 mM dNTPs and 1 U ribonuclease inhibitor RNasin. As a final step, the template-primer combination was added to the reaction mix on ice. Following an initia
    3. Reverse transcri.ptase polymerase chain reaction (RT-PCR)
    1. he MTT Lysis buffer (20% SDS , 50% Dimethyl formamide) and the O.D.s7onm was measured. The standard curve was plotted and the equation derived, used to calculate the number of metabolically viable cells in experimental groups. Percentage of viable cells was calculated by comparing the number of viable cells in treated wells with that of untreated wells
    2. MTT micromethod is a colorimetric assay based upon the conversion of the (yellow) MTT reagent (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide), a tetrazole to purple formazan in the mitochondria (succinate dehydrogenase) of the living cell which is quantified by measuring the optical density at 570nm. When the amount of purple formazan produced by untreated control cells is compared to that of treated cells, the effectiveness of the agent can be deduced through the production of a dose-response curve. Parasites in their log phase were harvested and the dead cells removed at 129 x g for 5min at RT. The live cells were resuspended in phenol red-free DMEM containing 10% FBS to a cell count of 2.5 X 107. 1mL of above culture was plated into each well of a 24 well plate and appropriate treatments were given for desired duration of time. Some untreated cells were also kept aside for generating the standard curve. After appropriate treatment, from each well of the 24 well plate 200pL was aliquotted into 4 wells of a 96 well plate. To each well of the 96 well plate, 10pL of MTT solution (5mg/ mL prepared in PBS and filtered with 0.22p filter) was added and the plate incubated at 23°C for 2-3 hours till colour develops in the control cells. A standard curve was also plated by taking different dilutions of untreated cells and processed similarly. Once colour developed, the reaction was stopped by lysing the cells using
    3. MTT assay to assess viability of Leishmania parasites
    4. Plasmid/Ligation mix was incubated with ultra-competent cells for 30 min on ice. This was followed by a heat shock at 42°C for exactly 45s after which the cells were chilled on ice for 2 min. lmL of LB (Luria Bertani) medium was added to the cells and incubated at 37°C in a shaker incubator for 45 min. Cells were then plated on LB agar plate containing appropriate antibiotic and/ or X-gal solution and incubated at 37°C overnight
    5. Transformation of competent cells with DNA