Lysis Buffer: 0.1% Triton X-100/0.1 M Tris-HCl (pH 8.0). 450 ml distilled water 50 ml 1M Tris-HCl (pH 8.0) 0.5 ml Triton X-100 detergent • 100X Mg++ solution: 0.1 M magnesium chloride 4.5 M 2-mercaptoethanol Stored at 4°C. • 0.1 M sodium phosphate (pH 7.5)41 ml 0.2 M Na2HPO4 9 ml 0.2 M Na H2PO4 50 ml distilled water • 4 mg/ml ONPG (o-nitrophenyl-β-D-galactopyranoside) in 0.1 M sodium phosphate (pH 7.5) containing 2 mM β-mercaptoethanol, Stored at –20°C. • 0.1 mg/ml β-gal standard: 0.1 mg/ml β-gal in 0.1 M sodium phosphate (pH 7.5) containing 2 mM 2-mercaptoethanol Stored at 4°C. • 1 M sodium carbonate in water

stored. To prepare competent cells pre-inoculum was prepared. A single bacterial colony was picked from LB agar plate, inoculated into 3 ml LB medium, and incubated overnight at 37°C temperature with shaking at 200 rpm. 1% of this pre-inoculums was sub cultured in 100 ml LB-broth and incubated at 18°C with shaking until OD at 600nm reached 0.5 - 0.6 (approx.). Culture was kept on ice for 10 min. with constant shaking.Cells were pelleted by centrifugation at 2000 g at 4°C for 8 min. Pellet was resuspended in 40 ml of ice-cold TB buffer. Bacterial suspension was kept on ice for 30 min, re-spun at 2000 g at 4°C for 8 min. Pellet was resuspended in 8 ml of TB buffer in which final concentration of DMSO was 7% and left on ice for 10 min. 100 μl aliquots were made and snap frozen in liquid nitrogen and stored at -80°C

All the salts (10 mM PIPES, 15 mM CaCl2.2H2O, 250 mM KCl, 55 mM MnCl2.2H2O) except MnCl2 were dissolved in milliQ water and pH was adjusted to 6.7 with 1N KOH. MnCl2 was dissolved separately in mill Q water. MnCl2 was added drop wise while stirring (MnCl2 if added directly will give a brown color to the solution and precipitate out, hence it needs to be dissolved separately). Solution was then filter sterilized and

argP+, argPd-S94L, argPd-P108S, argPd-P274Sfragment downstream of the phage T7-promoter, such that the encoded proteins beara C-terminal His6-tag provided by the vector DNA sequence. Theresultant plasmid was transformed into strain BL21(DE3) which has the T7 RNA Polymerase under the isopropyl thio-β-D-galactoside (IPTG) inducible lacUV5promoter.The resultant strains were grownin LB (500-1000 ml) to an A600of around 0.6and were then induced with 1 mM IPTG and harvested after 4-hrs of induction.Bacterial cells were recovered by centrifugation, resuspended in 20 ml of lysis buffer(20 mM Tris-Cl, pH-8; 300 mM NaCl; 10 mM DTT and 10 mM imidazole) containing20 μg/ml lysozyme, and lysed by sonication with 30-sec pulses for 10-min. Theprotocol for His6-ArgP(ArgPds)protein purification involved (i) passing the lysate through a 5ml Ni-NTA (Qiagen) chromatographic columnequilibrated with lysis buffer, (ii) washing thecolumn with 100 ml of washing buffer (20 mM Tris-Cl, pH-8; 300 mM NaCl; 10 mMDTT; 30 mM imidazole), and (iii) elution of His6-ArgP(ArgPds)from the column with elutionbuffer (20 mM Tris-Cl, pH-8;300 mM NaCl; 10 mM DTT and 250 mM imidazole) andcollection of 1.5 ml eluate fractions (10 fractions). The fractions were tested forprotein by Bradford method and the protein-carrying fractions (generally tubes 2 to 5)were pooled and dialysed in a 1:200 volume ratio against 20 mM Tris-Cl, pH-8 with 10mM DTT, 300 mMNaCl for 5 hrs followedby a change to buffer of composition 20 mM Tris-Cl, pH-8 with 10 mM DTT, 300 mM NaCl and 40% glycerol for 24 hrs. The proteins were concentrated by centrifugation toaround 1 mg/ml by using Amicon filter (pore size 10-KDa) and stored at −20ºC or −70ºC

For preparing ArgP and ArgPd-S94L, -P108S and -P274S proteins, derivatives(designated as pHYD1705, pHYD2678, pHYD2679 and pHYD2680 respectively) of the plasmidvector pET21b (Novagen) was constructed which carries the PCR-amplified

CAN is a toxic analog of Arg and is an inhibitor of bacterial growth. Strains were tested for sensitivity/resistance to CAN by streaking them on minimal A-glucose platessupplemented withoutand with40 μg/ml CAN(or other concentrations as indicated) and 40 μg/ml uracil

Competent cells for high efficiency transformations were prepared by a method ofInoue et al. (1990) with few modifications. An overnight culture of the strain (routinelyDH5α) was sub-cultured into fresh sterile LB-brothin 1:100 dilutions and grown at 18ºC to an A600of 0.55. The cells were harvested by centrifugation at 2500 rpm for 10-min at 4ºC. This was re-suspended in 0.4 volumes of INOUE buffer and incubated inice for 10 min. The cells were recovered by centrifugation at 2500 rpm at 4ºC for 10-min and finally re-suspended in 0.01 volume of the same buffer. Sterile DMSO wasadded to a final concentration of 7%. After incubating for 10-min in ice, the cells werealiquoted in 100 μl volumes, snap frozen in liquid nitrogen and stored at –70ºC

For routine plasmid transformations, following method which is modification of thatdescribed by Cohen et al. (1972) was used. An overnight culture of recipient strain wassub-cultured 1:100 in fresh LB medium and grown till mid-exponential phage. Theculture was chilled on ice for 15-min, and the steps thereafter were performed at 4ºC.20 ml of culture was centrifuged and pellet was re-suspended in 10 ml of 0.1 M CaCl2.After 15-min of incubation on ice, the cells were again centrifuged and re-suspended in2 ml of 0.1 M CaCl2. The suspension was incubated on ice for 30-min. To the 200 μl aliquot of the cell suspensionplasmid DNA (20 to 200 ng in less than 10 μl volume)was added, incubated for half an hron ice and given a heat shock for 90-sec at 41ºC.The cultures was rapidly chilled, mixed with 0.8 ml of LB-broth and incubated at 37ºCfor 1-hr, and plated on an appropriate selective medium at various dilutions. An aliquotof cell suspension to which plasmid DNA was not added served as a negative control

For SEM, C. glabratacells were fixed for 24 h in 2.5% glutaraldehyde in phosphate buffer (0.1 M, pH 7.2) at 4 ̊C, post-fixed in 2% aqueous osmium tetroxide for 4 h and dehydrated. After drying to critical point, mounted samples were coated with a thin layer of gold for 3 min using an automated sputter coater and visualized by SEM (JEOL-JSM 5600)

Yeast cell wall was isolatedas describedpreviously(De Groot et al., 2004). Briefly, cells grown underdifferent environmental conditions were harvested at 5,000 g for 5

Crude fractionation of total membraneswas carried outviadifferential centrifugation asdescribed previously (Moranoand Klionsky,1994)with slight modifications. Cells grown tolog-phase in YPDmedium werecollected, washed,normalizedto 10 OD600and resuspendedin 1 ml spheroplast buffer containing 1-2mg of zymolyase20T (MP Biomedicals).Following incubation at 30 ̊Cfor 30-45 min,spherolplastswerecollected by centrifugation at 800 g for 3 minat 4 ̊C and resuspendedin 1 mlice-cold Tris-EDTA (pH 7.5). Spheroplastswere lysed with 100 μl 0.5mm glass beads on a vortex mixer with 10 secpulsegiven thricewith intermittent ice-breaks.Cellsuspension was centrifuged at 800 g for 5 minat 4 ̊C to pellet unbrokenspheroplastsdown andthesupernatant was centrifuged at 15,000 g for 5 minat 4 ̊C to obtainthemembrane fraction pellet.Pellet was washed once with ice-cold Tris-EDTA (pH 7.5), resuspendedin 50 μl of the samebuffer and stored at -20 ̊Ctill further use. Protein concentration of pellet fraction was estimated using BCAprotein assay kit with BSA as thestandard

Trehalose from C. glabratacells was extracted by trichloro acetic acid (TCA)solutionas described previously (Lillie et al.,1980). Cells grown in YPDmediumwere collected at different time pointsof growth and washed thrice with ice-cold sterile water. Cells were immediatelystored at-20 ̊Ctill further use.For trehalose isolation, 10-20 OD600cells were thawed in 500 μl TCA (0.5 M) solutionon ice and incubated at room temperaturefor 1 h.Supernatant fraction was collected by sedimenting cells at 14,000 rpm for 5 minat 4 ̊C.TCA extractionwas repeated withcells once more and the resultingsupernatant was mixed with the earlier fraction.Extractedtrehalose was measuredby phenol-sulphuric acid methodof carbohydratedeterminationwithcommercially available purified trehalose(Becton, Dickinson and Co.) as a standard.Total trehalosecontent was normalized to the cell densityand expressed as μg/2 x 107cells

To assess the activity of plasma membrane proton pump, CgPma1, in cells grown in differentexternal pH environment,whole cell acidification assaywas carried out.This assay is a measurement of glucose-responsive proton pump activityin live cellsand is based on a decrease inthe pH of a weakly-buffered solutionupon extrusion of H+ions from thecell. The amount of change in the pH of the medium represents a crude measurement of the activity of functional plasma membrane proton pump in live cells. Whole cell acidification assay was conductedwithcellsgrown in YNB pH 5.5 and YNB pH 2.0medium as described previously (Martinez-Munoz and Kane, 2008) with slight modifications.After growth at30 ̊C for 2 h, cells were harvested, washed and resuspended(1.5-3.0 mg wet weight/ml) in 15ml MES/TEA (1mM; pH 5.0) buffer. Cell suspension was kept at 25 ̊C with continuousagitation. Extracellular pH of the buffer solution was recorded at 1 mininterval for 20 minwith the help of a pH meter(BT-600, BoecoGermany). To activate plasma membrane proton pumping, glucose and KCl were added to a final concentration of 40mM after 3 and 8 minincubation, respectively. Plasma membrane proton pump activitywas plotted as a change in the pH of the extracellular solutionversustime

Log-phase yeast cell cultures were harvested and total protein was extracted by lysingyeast cells using glass beads. Briefly,10 mllog-phase yeast culturesgrownin appropriate medium were harvested,washed once with ice-cold water and suspended in 250 μl homogenizing buffer containing 1 mM phenylmethylsulfonylfluoride(inhibitsserine proteases), 10 mM sodium fluoride(inhibit Ser/Thr and acid phosphatases), 1 mM sodium orthovanadate (inhibits Tyr and alkaline phosphatases) and 1X concentration of protease inhibitor cocktail(RocheCat # 04693159001). Cells were lysedwith glass beads by vortexing five times at high speed for 1 min with intermittent 1 min ice breaks. Unbroken cells and cell debris were removed by centrifugation at 1,000 g for 5 min at 4 ̊C. Cell lysate was collected and protein was quantified using bicinchoninic acid (BCA)protein assay kit (Thermo Scientific # 23227) as per supplier’s instructions

Themethod was used for isolation of good quality genomic DNA that wasused to map Tn7insertionin C. glabratamutants.Briefly,10 mlsaturated yeast culturewasharvested, resuspendedin 1 ml sterile water and transferred toa2 ml microcentrifuge tube. Cells were pelleteddown by centrifugation at 4,000 rpm for 5 min. Supernatant was discarded and the pellet was resuspendedin 500 μl freshly prepared solutioncontaining100mM EDTAand 5% β-mercaptoethanol andincubated at 42 ̊C for 10 min. After incubation,cells were spun down at 5,000 rpm for 1 minand resuspendedin 500μl freshly-prepared BufferB. One tip full of lyticase(Sigma # L4025) was added and cellsuspension was incubated at 37 ̊C for 1 h. Following incubation,cell suspension was spun down at 6,000 rpm to recover spheroplasts.Spheroplasts weregently resuspendedin 500μl BufferCand DNA was twice extracted with 500μl phenol:chloroform:isoamyl alcohol (25:24:1)solution.Aqueous layer was collected in a new 2ml microcentrifuge tube and DNA was precipitated with 1ml ethanol and 1/10thvolume of 3M sodium acetate (pH 5.2)by centrifugation at 13,000 rpm for 5 min. Pellet was resuspendedin 200 μl TE containing 0.3 μl of RNase Cocktail™and incubated at 37 ̊C for 30 min.After incubation, 300 μl additional TE was added and DNAwas re-precipitated withethanol and 3 M sodium acetateas described above. Pellet was washed with 70% ethanol anddried under air. DNA pellet was finally suspended in 100 μl TE and stored at -20 ̊C

Yeast cell viability was measured by plating appropriate dilutions of cell cultureonYPD plates at various time intervalsduringgrowth.Cell suspension was diluted in1X PBS. YPD plates were incubated at 30 ̊C for 2-3 daysand total colony forming units(CFUs)were calculated by counting the number of coloniesthat appeared onYPDplatesand dividing that number by anappropriate dilution factor

E. colistrains carrying plasmids were inoculated and grown overnight at 37ºC and 200 rpm in LB-liquid medium supplemented with either 50 μg/ml ampicillinor 30 μg/ml kanamycin. Cells were harvested by centrifugation at 2,500g for 5 min. Plasmids were extracted using Qiagen plasmid miniprep kit following the manufacturer’s instructions. Concentration of the extracted plasmid DNAs was measured using spectrophotometerat 280 nmandstored at -20ºC

weretransferred toa sterile 100 mm cell culture dishcontaining 11 mlfresh and prewarmed completemedium andculturedin tissue culture incubatorat37°C and 5% CO2.After 12hincubation, medium was replacedwith fresh prewarmed mediumand cells were allowed to proliferate till they acquire 80% confluence

Freezer stocks of THP-1 and Lec-2 cells were prepared either in commercial cell preservation medium (Gibco) or completemedium supplemented with 10%heat inactivated serum and 10% DMSO. For cryopreservation, 5-6 million cells were resuspended in 0.5 mlfreezing medium in 2 ml cryopreservation vials,stored in an isopropanol bath and were transferred to-70°C freezer. Aftertwo days, freezer stocks were transferred to liquid nitrogen containertill further use. To revive the cells, freezer stocks were taken outfrom liquid nitrogen container and transferred immediately to37°C water bath. After2-3 min, when freezing medium hadthawed completely,cells

Overnight grown bacterial culture (3ml)was pellet down by centrifugationat4ºC for10-min at 6000 rpm. The cells were re-suspended in 200μl of Resuspension solution(solutionI). 400μl of freshly prepared Lysissolution(solution II)was then added and mixed by gently inverting the tubesfor 4-6 times and allowed to lyse for 5 min at room temperature.The complete lysis was ascertained by uniformity and clarityof the contents. Subsequently, 400μl of Neutralization solution(solution III)was added and the tubes were inverted 4-6 timesand gently for homogeneous mixing followed byincubation for 5 min on ice. After centrifuging at 12,000 rpm for 15-min, supernatant was decanted into a fresh tube, and0.7 volume of iso-propanol was added.Theprecipitated nucleic acids were then recovered by centrifugation at 12,000 rpm for 30-min. The pellet was washed once with 70% ethanol, air-dried and re-suspended in 100μl of TE-buffer. It was treated with RNase at a concentration of 20μg/ml by incubating at 37ºC for 1hour. It was further extracted with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) mixture. After centrifugation, the clear supernatant was used for recovering the nucleic acids. The nucleic acids were precipitated with 2.5 volumesof ethanolin presence of3 M sodium acetate. In case where high purity plasmid preparations are required (for transfection to cells) the plasmid isolation was carried out with the commercially available midiprep or miniprep kits following the manufacturer’s instruction. Plasmids were observed on 1% agarose gel

drop wiseaddition and kept at 4⁰C for 24h. Cells were then washed with PBS and stained with DNA staining solutionat 370C in darkwith intermittent shaking. The DNA content of cells was measured by flow cytometryusing FACS-Aria (Beckton-Dickenson) at 695 ±20 nm using a 655 long pass filter.The DNA content was then analysed by FACSDivaor FlowJosoftwareto evaluate the various phases of cell cycle. The diploid 2N DNA content was referred as G1/G0 population and the 4N DNA content was referred as G2/M population. Cells with intermediary DNA content (between 2N -4N) content were considered as S phasecells and those below 2N DNA content as sub G0 cells

Thecells were collected at various time points by trypsinization, washed in phosphate buffered saline (PBS, pH 7.2) and fixed in chilled 70% methanol: ethanol (1:1) solution by

development. Absorbance was measured at 490 nm, and concentration of glucose production was calculated against glucose standard. Cellulase activity is expressed as micromoles of reducing sugar (glucose) released per minute per 109cells. For plate assay, cell-free culture supernatant of X. oryzaepv. oryzaestrains were inoculated in wells of 0.2% CMC agarose plates. In addition, cellulase assay was also performed by spotting the colony on 0.2% CMC PSA plates. Plates were incubated for 8 to 24 h and stained with congo red to observe the halo formation as described previously (Wood and Bhat, 1988). Extracellular xylanase activity in different X. oryzaepv. oryzae strains was measured using 0.2% 4-O-methyl-D-glucurono-D-xylanremazol Brilliant Blue R (RBB-Xylan) (Sigma-Aldrich) as substrate (Biely et al., 1988)on 1% agarose plates. Xylanase activity is indicated by production of halo around the bacterial colony (Ray et al., 2000). Similarly, for lipase activity p-nitrophenyl butyrate was used as substrate. Lipase activity was calculated by measuring the level of p-nitrophenol released upon hydrolysis of p-nitrophenyl butyrate at 410 nm (Acharya and Rao, 2002). Lipase activity was expressed as micromoles of p-nitrophenol released permin per109cells. For plate assay, colonies were spotted on 1% PSA plates containing 0.5% Tributyrin in 100 mM Tris (pH 8) and 25 mM CaCl2 and halo formation was observed for lipase activity

For extracellular enzyme assays, X. oryzaepv. oryzae strains were grown in PS, MM9 and XOM2 media to an OD of 0.6, and centrifuged at 12,000 g for 10 min to collect the supernatant. The supernatant was taken as an extracellular fraction and cell pellet was plated by dilutionplating to get the CFUs per milliliter of culture. Extracellular cellulase activity was measured using phenol-sulphuric acid (H2SO4) method, which measures pentoses and hexoses (concentration of glucose released) upon cellulase activity (DuBois et al., 1956). Briefly, a specific amount of supernatant was taken and volume was adjusted to 300 μl by adding 50 mM acetate buffer (pH-5.4). To this, 1% carboxy methyl cellulose (CMC) substrate solution was added and mixed well. This mixture was incubated at 28°C for 30 min, and the reaction was stopped by adding 1 ml ice-cold ethanol. Solution was mixed well, kept on ice for 5 min and centrifuged at 12,000 g for 5 min. Supernatant was recovered and 5% phenol was added to it, mixed well followed by adding 1 ml H2SO4. The tube was incubated at RT for 20 min for co

Xanthomonas strains were grown in PS medium for 14-16 h at 28°C with continuous shaking at 200 rpm. 1 ml of bacterial cultures were ten-fold serially diluted in water and 100 μl volume of each dilution was plated on PS agar plates to get the colony forming units (CFUs). Similarly, 5 μl volume of each dilution was spotted on PS agar plates containing different concentration of streptonigrin and different detergents for intracellular iron and membrane sensitivity assay, respectively. Plates were incubated at 28°C and images were captured after 2-8 days of incubation depending upon m

This method was used to isolate highly pure genomic DNA. Briefly, 10 ml overnight grownC. glabratacultures were spun downandwashed with 10 ml sterile water. Washed cells wereresuspended in500 μl sterile water and transferred toa1.5 ml microcentrifuge tube. Tubes were spundownat 4,000 rpm for 5 min, supernatant was discarded andcell pellet was resuspended in 500 μl of buffer containing 100 mM EDTA and 5% β-mercaptoethanol and incubatedat 42°C for 10 min. Post incubation, cells were spun down at 4,000 rpm for 5 min and resuspended in freshly prepared Buffer B. To this, one tip-full of lyticase (Sigma, L4025) was added and incubated at 37°C for 1 h.After incubation, spheroplasts were collected by spinning downtubes at 6,000 rpm for 5 min, supernatant was discarded and the pellet was resuspended in 500 μl of Buffer C. DNA was extracted twice with 500 μl of PCI (25:24:1) solution and the aqueous layer was transferred toa new1.5 ml microcentrifuge tube. To this, 2.5 volume of absolute ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.3) wereadded. Tubes were spundownat 13,000 rpm for 10 min, DNA pellet was resuspended in 200 μl of 1X TE buffer containing0.3 μl of RNase cocktail (Ambion) and incubated at 37°C for30 min. DNA was precipitated again by adding absolute ethanol and sodium acetate as mentioned above. DNA pellet was washed once with 70% ethanol, centrifuged at 13,000 rpm for 10 min, air-dried at room temperature and was resuspended in 100-200 μl of 1X TE buffer by gently tapping the tube. DNAwas stored at -20°C until use

This method was used to isolate highly pure genomic DNA. Briefly, 10 ml overnight grownC. glabratacultures were spun downandwashed with 10 ml sterile water. Washed cells wereresuspended in500 μl sterile water and transferred toa1.5 ml microcentrifuge tube. Tubes were spundownat 4,000 rpm for 5 min, supernatant was discarded andcell pellet was resuspended in 500 μl of buffer containing 100 mM EDTA and 5% β-mercaptoethanol and incubatedat 42°C for 10 min. Post incubation, cells were spun down at 4,000 rpm for 5 min and resuspended in freshly prepared Buffer B. To this, one tip-full of lyticase (Sigma, L4025) was added and incubated at 37°C for 1 h.After incubation, spheroplasts were collected by spinning downtubes at 6,000 rpm for 5 min, supernatant was discarded and the pellet was resuspended in 500 μl of Buffer C. DNA was extracted twice with 500 μl of PCI (25:24:1) solution and the aqueous layer was transferred toa new1.5 ml microcentrifuge tube. To this, 2.5 volume of absolute ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.3) wereadded. Tubes were spundownat 13,000 rpm for 10 min, DNA pellet was resuspended in 200 μl of 1X TE buffer containing0.3 μl of RNase cocktail (Ambion) and incubated at 37°C for30 min. DNA was precipitated again by adding absolute ethanol and sodium acetate as mentioned above. DNA pellet was washed once with 70% ethanol, centrifuged at 13,000 rpm for 10 min, air-dried at room temperature and was resuspended in 100-200 μl of 1X TE buffer by gently tapping the tube. DNAwas stored at -20°C until use

Yeast genomic DNA was isolated by mechanically lysing the yeast cells. Briefly, 10 ml of overnight grown yeast culture was transferred toa 15 ml centrifuge tube andcells were spun down at 4,000 rpm for 5 min. Media was decanted and cells were washed with 10 ml sterile water. Washedcells were resuspended in 500 μl of Buffer A and transferred to a 1.5 ml microcentrifuge tube. Tubes were incubated at 65°C for 15 min. Post incubation,500 μl of PCI (25:24:1) solution was added. To this, 0.5 g of 0.5 mm glass beads were added and cells were lysed mechanically in a bead-beatinghomogenizer (MP Biomedicals,FastPrep®-24) thrice, 45 seceach, with intermittent cooling on ice. Tubes were spun at 12,000 rpm for 5 min and the aqueous layer was transferred to a new 1.5 ml microcentrifuge tube. To this, 500 μl of PCI solution was addedand mixed gently by inverting the tubes.Tubes were centrifuged again at 12,000 rpm for 5 min and aqueous layer was transferred to another 1.5 ml microcentrifuge tube. Next, 2.5 volume of absolute ethanol was added to the aqueous layer, mixed well and centrifuged at 13,000 rpm for 10 min. Supernatant was decanted and the DNA pellet was washed once with 70% ethanol and centrifuged at 13,000 rpm for 10min. Washed DNA pellet was air-dried and dissolved in 100-200 μl of 1X TE buffer by gently tapping the tubes

phorbol myristateacetate (PMA) (Tsuchiya et al., 1982). For PMA treatment, THP-1 cells were allowed to grow till 70-80% confluence and were collectedin a centrifuge tube by centrifugationat 1,000 rpm for 3 min. THP-1 cell pelletswere resuspended in 4-5 ml of pre-warmed complete RPMI-1640 medium, 100 μl of this cell suspension was appropriately diluted in PBS (1X) and viability was determined by counting trypan blue stained cellsusing hemocytometer. THP-1 cell suspension was diluted appropriately to obtainafinal cell density of 106cells/ml with pre-warmed complete RPMI-1640 medium. PMA was added totheTHP-1 cell suspension at a final concentration of 16 nM and mixed well by gently inverting the tubes. PMA-treated cells were seeded either in 24-well cell culture plates or in cell culture dishes and allowed to grow for 12 h under tissue culture conditions i.e. at 37°C and 5% CO2.After 12 h incubation, spent medium was replaced withfresh pre-warmed complete RPMI-1640 medium and cells were allowed to recover for another 12 h

THP-1 monocytes getdifferentiated intophagocytic macrophages upon treatment with