The overall goal of this procedure is to establish an in vitro culture model system to study intracellular behavior of a human opportunistic fungal pathogen. Canida lab ratta in macrophages derived from human monocytic cell line TP one. To achieve this THP one monocytes are first treated with four ester to differentiate them into macrophages.
Next THP one macrophages are infected with canida GTA cells two hours post-infection. Non phagocytose canida GTA cells are removed by PBS washers and intracellular e cells are recovered for further studies by osmos of infected macrophages. This protocol can be used to assess both the viral to canida GTA cells in macrophages as well as the faiz maturation Ross and cytokine response in THP on macro FIDS upon candida GTA infection.
In addition, this protocol can be scaled up to screen the candida collaborator mutant library for altered survival profiles in TSP one macrophages in a semi high throughput manner. The main advantage of this protocol is that it is relatively inexpensive, highly reproducible, and easily adaptable to different immune cells As well as fungal pathogens. To Begin the Procedure two days prior to infection, brought THP and monocytic cells to 80%CO on day one, take out the culture risk and slowly, gently to bring all subtle cells into suspension.
Transfer the DHP one monocyte cell suspension into a 15 ml centrifuge tube. Centrifuge The cell suspension at room temperature For four minutes At 1000 RPM Deccan the Medium into a waste container at five ml pre API medium into the tube and reus. Suspend the THV one cell pellet by gently pipetting the medium three four times.
Take 10 Microliter of the cell suspension to determine the cell density using a hemo cytometer and adjust the cell density to 1 million cells per ml. Add one microliter of one 60 micromolar PMA stock solution to the cell suspension so that the final concentration is 16 ano molar and mix it well. Dispense one ml cell suspension corresponding to 1 million cells into each well of a 24 well tissue culture plate and transfer it into an incubator set at 37 degrees Celsius and 5%CO2.
After 12 hours of incubation, discard the old medium. Add one ml pre RPI medium to each well and incubate the plate for another 12 hour. These cells will be used for Canada atory Infection, which will be shown later.
Using proper microbiological practices inoculate a single EA colony into a flass containing ML YPD medium. Incubate the flass for 14 to 16 hours in an incubator set at 30 Degrees Celsius following incubation centrifuge. One ML Culture at 4, 000 RPM for five minutes.
Discard the sup natin and wash shells three times with sterile PBS. After discarding the wash buffer, suspend the cell palette in one ml sterile PBS by gentle pipetting. Next, measure absorbance of the cell suspension in a spectro photometer at 600 nanometer and adjust the cell density to 2 million cells per ml with PBS.
Take 50 microliter of the cell suspension and dilute a hundred fold In PBS spread plate a hundred microliter culture on YPD AGA medium. Incubate the plate at 30 degrees Celsius and count the number of colonies that appeared on YPD plate after one or two days, multiply this number with appropriate dilution factor to calculate zero hour colony forming units. These many colony forming units of Canada Reta will be used to infect DHE on macrophages.
In the next Step, add 50 macro Liter of Canada clater cell suspension to macrophage mono layer in a 24 whale culture plate. Soil the plate to evenly distribute each cells and incubate at 37 degrees Celsius after two hour incubation. Discard the medium by inverting the culture Plate into a container to each well of the culture Plate at ml serial PBS carefully along the side of the well wells thoroughly and Discard the buffer.
Repeat this step twice more. Next except for the First well add one ML RPI medium to all other wells, which can be used to monitor intracellular replication of candida atory cells at 4, 6, 12, and 24 hours post infection to recover intracellular e cells after two hours of macrophage infection, add one ML cereal water to the phos. Well after two minutes, scrape the well gently with the one ML piper tip to remove The macrophage debris of the well.
Rinse the well and collect the ly it in a macro centri fish tube. Prepare a hundred fold dilution of microFIT cell lysate in PBS and plate a hundred microliter on YPD AGA medium. Incubate the plate at 30 degrees Celsius for one to two days and count the number of colony forming units.
Multiply this number with appropriate dilution factor to enumerate the number of canida ator cells that are phagocytose by T two and macrophages after two hours infection. Similarly, the number of intracellular canida ator cells can be determined at different time points. Post infection and intracellular replication can be represented as an increase in the at 24 hours compared to the CFUs internalized at two hours shown here.
Other results of a typical experiment wherein the total number of canida gator cells infected to TV one macrophages is six times 10, two per four. The number of cells recovered at two hour is 3.7, six times 10.4. Thus illustrating that e cells are phagocytosed at a rate of 62.66%during the 24 hour co incubation period.
Can al cells other than a 5.2 fold replication as the number of intracellular e cells increased from 3.76 times 10.4 to 1.96 times 10.5. In this experiment, intracellular replication of canida TER cells can also be assessed using GFP expressing E cells while confocal microscope for this. Prepare and infect THV and macrophages in a four chamber slide using the protocol described earlier for the 24 well culture plate.
Two hours post-infection. Discard the medium by inverting the slide into a waste container at 500 microliter PBS to each chamber of the slide. Rinse well and discard PBS.
Repeat this step two more Times at 500 microliter of 3.7%formal height to fix the cells. Incubate the slide at room temperature for 20 minutes and discard formal de Height. Wash each well price with 500 microliter PBS to perme mobilize Macrophages at 500 microliter triton X after five minutes incubation, remove Triton X wash the well strike with PBS.
Carefully disassemble The chamber slide using a chamber removal and air rider slide. Place one drop of vector Shield mounting medium containing DPI on each sector of the slide mount a cover slip and seal the edges with nail paint visualized cells under a confocal microscope. Shown here are the representative confocal images of TP one macrophages infected with GFE expressing wild type canida laboratory cells about a sixfold increase in the number of intracellular e cells from two to 24 hours is indicative of canida laboratory replication in TP one macrophages signature tag mutagenesis approach is widely used to identify ulence factors in microbial pathogens wherein large number of mutants are simultaneously screened for growth defects via unique oligonucleotide sequence tag.
Here we describe our protocol for the STM screen of Canada Library and mutant library, which consists of following three steps. First 96 plasmids. Each harboring a unique oligonucleotide sequence stack are immobilized on a nylon membrane.
In the second step, a pool of 96 CANIDA ator mutants is screened for altered survival profiles in THV one macrophages, followed by recovery of YPD, grown and macrophage internalized mutant e cells, which will be referred as input and output samples respectively. Lastly, DNA signature tags are PCI amplified and radio label from input and output genomic DNA samples. The PCR product is hybridized to unique tags and radioactive signals on the membrane are quantified and analyzed.
To assemble the 96 12 dot board apparatus, place a wet nylon membrane on top of a moist swap wind tree paper and tighten the screws of the unit. Wash the membrane with sterile water, followed by a wash with 0.4 mu sodium hydroxide. Transfer 200 nanogram of PLA BDNA solution from the 96 12 block to the corresponding wells of the dot blood apptus Following a 0.4 molar sodium Hydroxide wash.
Remove the membrane from the dot blood apparatus after a ference in two XXSC crossing the transferred plasma BDNA to the membrane in a UV crosslinker. Incubate The membrane's in antennal pre hybridization, buffer and input and UNE bottles for two hours at 42 degrees Celsius. Prepare radio level probes by PCR using genomic DNA extracted from input and output cell palettes and primers against the invert flanking region of unique Tags.
Denature the radio label probes at 99 Degrees Celsius for five minutes and snap gel on ice. Transfer the de probes to input and output bottles and incubate for 14 to 16 hours at 42 degrees Celsius. Post hybridization.
Wash the input and output membranes as described in the protocol. Take out the membranes from the bottles air dry and expose them to a phospho images screen. After two to three hours, scan the phosphorus screen and acquire images.
In this example, two hybridized membrane filters are shown, which represents screening of one pool of 96 canida Gatory mutants for altered survival. In thv one macrophages quantification and determination of the ratio of signal intensity of each spot on the output to the input membrane revealed that tag numbers 13 26 75 and 85 are underrepresented in the output. These stacks are encircled in red and reflect the growth of four different mutants.
In contrast, mutants stack with unique sequences 11 and 81 highlighted in green are overrepresented in the output sample. In other words from this screen, pool of 96 mutants two mutants should increase survival while four mutants display at growth in macrophages. The altered survival phenotypes identified in such a pool screen should be verified in single infection assays.
After watching this video, you should have a good understanding of how to enact and in vitro oral system to study intra growth profile of the fungal pathogen of your interest. In addition, you should be able to conduct large scale mutant pool screening using the system now.
