This protocol helps verify the existence of Wolbachia in cells, which is the basis of understanding the interaction between Wolbachia and its host. In this protocol, four methods were used to successfully detect intracellular Wolbachia infection, which corroborated and improved the detection accuracy of Wolbachia infection of cells. Demonstrating the procedure will be Qiuqiu Xiao, a postgraduate from the Key Laboratory of Modern Pathogen Biology and Characteristics.
Begin by observing unstained Aa23 and Aa23-T cells at 100x magnification using light microscopy. Culture the cells for five to seven days to reach 70 to 80%confluence per flask. Using a pipette, transfer 1 mL of the culture medium to a microcentrifuge tube.
Centrifuge at 100 RCF for five minutes to harvest the cells. Remove the supernatant and store the resulting cell pellet at minus 20 degrees Celsius. Culture the cells for five to seven days to reach 90 to 100%confluence per flask.
Resuspend the pellets in 0.40 mL of PBS and transfer 50 microliters of this solution to a microcentrifuge tube. Add five microliters of 20 mg/mL proteinase K and incubate at 55 degrees Celsius for one hour. Then incubate the tubes at 99 degrees Celsius for 15 minutes to obtain the crude DNA and store it at minus 20 degrees Celsius.
To perform PCR, prepare a total reaction mixture of 20 microliters and set the PCR conditions as described in the text manuscript. Detect the DNA on a 1%agarose gel using a gel imager. Perform quantitative PCR amplification using TB Green in a real-time PCR system and record the results of each reaction.
Analyze the DNA in a total reaction volume of 20 microliters and set the quantitative PCR conditions as described in the text manuscript. Calculate the WSP and RPS6 gene copy numbers in the cells according to the established standard curve. Then calculate the Wolbachia relative density by the copy number of wsp/RPS6.
Analyze the data using an independent samples t-test. Add 200 microliters of lysis buffer to the cell pellet and incubate on ice for 30 minutes. Centrifuge the lysates at 12, 000 RCF for 10 minutes at four degrees Celsius and aspirate the supernatant.
Analyze the wsp concentration of the supernatant using a BCA protein assay kit, following the manufacturer's instructions. Load equal amounts of protein on a 15%SDS-PAGE gel. Transfer the protein to nitrocellulose membranes and block the membranes with 5%skimmed milk for two hours at room temperature.
Then wash the membranes three times with TBST. Incubate the membranes overnight at 4 degrees Celsius with 100 microliters of anti-wsp polyclonal antibody prepared by diluting the primary antibody with 5%skimmed milk. Wash the primary antibody three times with TBST.
Add 100 microliters of horseradish peroxidase-conjugated secondary antibody to the membranes and incubate it on a shaker for one hour at room temperature. Wash the membranes thrice with TBST and mix 20 microliters of solution A and 20 microliters of solution B in the chemiluminescence detection kit at a ratio of one to one. Immerse the entire membrane into the luminescence solution simultaneously and observe the signals using a multifunctional imaging system.
On a Laser confocal Petri dish, incubate Aa23 and Aa23-T cells for three to four days at 28 degrees Celsius under 5%atmospheric carbon dioxide. When the cell reaches 60 to 70%confluence, wash the cells three times with PBS. Fix the cells in one mL of 4%paraformaldehyde for 20 minutes at four degrees Celsius.
Wash the fixed cells using PBST for five minutes and rewash the cells twice with PBS. Incubate the Petri dish in one mL of 3%bovine serum albumin for one hour at 37 degrees Celsius. Add 100 microliters of mouse anti-wsp polyclonal antibody and mouse serum to the slides and incubate it overnight in a wet box at four degrees Celsius.
Remove the Petri dish from the wet box and return them to room temperature. Wash them three times with PBS and remove the PBS. Protect the following procedures from light.
Incubate the Petri dish with 100 microliters of an Alexa Fluor 488-conjugated goat anti-mouse antibody at 37 degrees Celsius for 30 minutes. Incubate the sample Petri dish with 50 microliters of DAPI diluted in PBS to 10 micrograms/mL at 37 degrees Celsius for five minutes and then wash them three times with PBS. Observe the immunostaining under a confocal microscope.
Before detecting Wolbachia, Aa23 and Aa23-T cells were observed under a light microscope to determine morphological differences between the two cell lines. The Aa23 and AA23-T cells had at least two cell morphologies but no apparent morphological differences were observed between the two cell types. Using the diagnostic primers 81F/691R, positive amplification of the Wolbachia wsp gene was detected in Aa23 cells but not in Aa23-T cells.
The analysis of Wolbachia density in the cell lines showed a wsp/rps6 ratio of 2.4 in Aa23 cells but no Wolbachia in Aa23-T cells. Western blot analysis of protein extracts showed a strong wsp signal for Aa23 but no signal was detected for Aa23-T. The indirect immunofluorescence assay detected the wsp protein in Aa23 cells, while only DAPI-stained DNA was detected in Aa23-T cells.
Ensure that the culture flasks are incubated at 28 degrees Celsius under 5%atmospheric carbon dioxide for five to seven days. This procedure can be followed by electron microscopy where one needs to pay particular attention to sample preparation and sectioning. The four experimental methods used in this study confirmed the detection accuracy of Wolbachia infection in cells, which is also applicable to other types of cells and tissues.
