Immunology and Infection
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Analyses of Proteinuria, Renal Infiltration of Leukocytes, and Renal Deposition of Proteins in Lupus-prone MRL/lpr Mice
Chapters
Summary June 8th, 2022
The present protocol describes a method to track lupus progression in mice. Two additional procedures are presented to characterize lupus nephritis based on cell infiltration and protein deposition in the kidneys.
Transcript
These methods can help investigate the progression of chronic inflammation associated with kidneys in lupus patients. We are offering a reliable and cost effective alternative that can be used to monitor the disease course in lupus. Begin sterilizing the hood by spraying 70%ethanol and place a sterile plastic sheet on the surface.
Then prepare tips, 1.5 milliliter tubes and a pipette to collect about 20 microliters of the urine. Take the cage and spray 70%ethanol before placing it inside the hood. Afterward, hold the mouse with one hand and use the other hand to gently massage the ventral area from top to bottom.
Using a 1.5 milliliter tube or pipette, collect the urine directly. Or if the sample drops collect it from the plastic sheet. Then put the mouse back in the cage.
Cut the dissected kidneys into grain size and digest in five milliliters of digestion buffer containing Collagenase and DNase 1 in RPMI 1640 medium containing HEPES for one hour with continuous gentle shaking at 37 degrees Celsius. Add 10 milliliters of ice cold PBS containing 10 millimolar of EDTA and incubate for 10 minutes on ice. Then vortex the suspension twice.
Later, filter the cell suspension through a 100 micrometer strainer and wash with 10 milliliters of HBSS full. Afterward, centrifuge at 350 times G for 10 minutes at room temperature. Prepare a 30, 37 and 70%stock isotonic Percoll solution and add a layer of 37%Percoll solution on top of the 70%solution.
Re suspend the cells in five milliliters of 30%stock isotonic Percoll. Using a Pasteur pipette slowly load 37%of five milliliters top and 70%of five milliliters on the bottom, making stock isotonic Percoll gradient. Then, centrifuge with up number nine and down number zero at 1000 times G for 30 minutes at room temperature.
Afterward, collect leukocytes from the 37 to 70%interface and re suspend them in five milliliters of C 10. Again, centrifuge at 350 times G for 10 minutes at four degrees Celsius. Subsequently re suspend the cell pellet in three milliliters of fax buffer and centrifuge at 350 times G for five minutes at four degrees Celsius.
Then discard the supernatant leaving about 50 microliters of the total volume. Add 50 microliters of FC receptor block per tube. Following this mix and incubate on ice in the dark for 10 minutes.
Later, add two milliliters of fax buffer for washing. Then, centrifuge at 350 times G for five minutes at four degrees Celsius and discard the supernatent leaving about 50 microliters of the total volume. Next, add 20 microliters of the appropriate fluorescent dye and let it stand for 15 to 30 minutes on ice.
Add 50 microliters of antibody mix per tube containing CD 138 brilliant violet seven 11 and CD 45 Alexa Fluor 700 and incubate on ice in the dark for 15 to 30 minutes. Afterward, add three milliliters of fax buffer. Centrifuge at 350 times G for five minutes at four degrees Celsius.
Remove the supernatant by vacuum leaving about 50 microliters of the total volume. Later, re suspend the cell pellet in 200 microliters of PBS. To collect the sample embed the half kidney in the small plastic cryomold with the optimal cutting temperature compound.
Then add dry ice into a polystyrene foam box and carefully place the cryomold on top of the dry ice. Next, remove the cryomold, wrap it in aluminum foil and store it at minus 80 degrees Celsius until further use. Then fix the dry to four micrometer sections with cold acetone for 10 minutes.
After fixing the slides, air dry for 0.5 to one hour and store them at minus 20 degrees Celsius for a short time or minus 80 degrees Celsius for a long time. To stain the samples, refix the slides in cold acetone for five to 10 minutes and allow the sections to warm up to room temperature. Afterward, using a pat pen, draw a hydrophobic circle around the section and allow it to dry.
Then place slides in TBS in the Copeland jar for 20 minutes and shake gently by putting the jar on the shaker. Subsequently, pour off TBS and fill the jar with TBS 5%BSA and 0.1%TW 20. Later, incubate the jar for 20 minutes by shaking gently on the shaker.
Then, take the slides out and wipe the bottom of the slides. Add 100 microliters of conjugated antibodies C3 fit C and IgG2 APE to the section and incubate room temperature in a humidified chamber for one hour. Wash the section thrice in PBS 5%BSA and 0.1%TW 20 for 10 minutes on the shaker and then shake dry the slides.
Mount the section with an anti-fade reagent and then with a cover slip. Then store the slides at four degrees Celsius until viewing under the microscope. For image analysis using the image J software, outline the desired region.
Afterward, set standard parameters by clicking on analyze then set measurements and measure area to get the results. Next, transfer the data obtained from the measure windows to a spreadsheet. Once done, click on analyze to measure the region and transfer the data again to the previous spreadsheet.
The protein levels in the urine of MRL LPR mice were detected over time where they were treated with phosphate buffered saline as the control group and lactobacillus reuteri as the treatment group. The mice group treated with L.reuteri at a more aggressive proteinuria progression than the control group. The fax analysis was performed for the isolated kidney leukocytes to analyze the plasma cells.
Further, the mice were treated with vancomycin or vancomycin along with Escherichia coli double stranded DNA. Although Escherichia coli double stranded DNA was expected to trigger the renal infiltration, no significant difference was found in the percentage of plasma cells. The complement C3 and IgG2a were detected through immunofluorescence staining on female MRL IPR kidney sections.
The effective environmental factors on the renal deposition of C3 and IgG2a was compared for the in-house mice bread in the animal facility and those purchased from a vendor. The immunohistochemical analysis did not show any difference between the two groups. Adding each layer of the percentage stock ISO 20 Percoll solution can be challenging, and if they mix you have to start over again.
This procedure helps with the flow cytometer and in vitter experiments, so you cannot study the different kidney cell type of populations. This procedure allows us to study the B-cells infiltrates in the kidneys and there is not a lot of information in the literature.
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