The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

The overall goal of this procedure is to characterize the expansion of T follicular helper cells, germinal center, B cells, and plasma cells by flow cytometry in the BM 12 model of systemic lupus erythema ptosis, or SLE. This method can help answer key questions in the lupus field, such as whether targeting particular molecular pathways with therapeutics may halt or slow disease progression. The main advantage of this SLE model is that it utilizes mice on a pure CPD seven black six background, so the researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time.

After isolating and injecting BM 12 donor cells into mice, determining the efficiency of grafting and euthanizing the mice according to the text protocol, lightly spray 70%ethanol to wet the abdomen of the mouse with surgical scissors. Make a small superficial incision approximately one centimeter above the genitalia, draw back the skin of the abdomen towards the sternum while keeping the peritoneal fascia intact. Fit a five milliliter syringe with an 18 gauge needle with the needle directed up towards the animal's head and add a 15 degree angle to the plane of the fascia.

Insert the needle into the lower right quadrant of the abdomen. Position the needle near the tip of the cecum to help prevent the needle from getting clogged with intestine while aspirating ascites. Next, carefully rotate the mouse on its side.

Then slowly draw ascites into the syringe. Once ascites have been recovered, move the syringe and record the aspirate volume from the markings on the side of the syringe. Discharge ascites into a five milliliter round bottom tube and store on ice for later processing to harvest blood via.

Draw from the vena cava or IVC as previously published. Use doll forceps to gently move the intestines to the left side of the mouse, uncovering the IVC. Insert a 27.5 gauge needle fitted to a one milliliter syringe into the IVC and slowly draw 400 to 500 microliters of blood to minimize hemolysis.

Slowly inject blood into a 0.5 milliliter micro centrifuge, serum, or plasma collection tube to dissect additional relevant tissues. Gently place the intestines back toward the right side of the animal and pull on the pancreas to remove the spleen. Remove any pancreatic tissue or splenic lymph nodes which remain attached.

Then immediately after dissection, use a high precision balance to weigh the spleen as gross s splenomegaly is a commonly reported parameter. In mouse models of SLE, place lymphoid tissues into individual 1.5 milliliter tubes filled with one milliliter of complete medium on ice. Within two hours of collection, centrifuge the blood at 10, 000 times G and four degrees Celsius for three minutes.

Remove the serum and Ali quad 10 to 20 microliters per tube to minimize freeze thaw cycles. Store at negative 80 degrees Celsius for later analysis of a NA by Eliza Next centrifuge. The ascites at 400 times G for five minutes.

With a one milliliter pipette, remove the S supernatant and Eloqua into several 0.5 milliliter tubes. Freeze at negative 80 degrees Celsius for later analysis. Re suspend the cellular fraction in approximately one milliliter of complete medium and transfer 200 microliters to a 96 well U bottom plate.

For flow staining. Mash each spleen through 70 micrometer cell strainers into separate tubes and use complete medium torin centrifuge at 400 times G for five minutes before decanting. The sup natant with one milliliter of cold RBC lysis buffer resus.

Suspend the cytes for one minute and mix by gently rocking using cold complete medium. Bring the volume up to 10 milliliters and centrifuge at 400 times G for five minutes before decanting The supra natant use five milliliters of complete medium to resuspend the cell pellet and with a hemo cytometer count the cells, adjust the volume so that 200 microliters contains one to 3 million cells. To stain for flow cytometry.

Transfer one to 3 million spleen cytes in complete medium into individual five milliliter round bottom tubes or into separate wells of a 96 well U bottom plate. Centrifuge the plate at 500 times G and four degrees Celsius for three minutes. Then flick the snat from the plate into an appropriate biohazard container To resuspend the cell pellets.

Use 100 microliters of flow buffer containing a fixable viability dye at the manufacturer's recommended concentration and purified anti CD 16, anti CD 32 antibody cocktail at one microgram per milliliter. Incubate the cells at 20 to 25 degrees Celsius for 10 minutes protected from light, and then add 100 microliters of cold complete medium to quench the viability die centrifuge the plate at 500 times G for three minutes. Then discard the SUP natant into the biohazard container.

Reese, suspend the cells in 200 microliters of flow buffer and centrifuge again before discarding the S supernatant to quantify donor CD four T-cell and recipient B-cell differentiation and expansion. Use an extended antibody panel as shown here and stain cells for flow cytometry. Using 50 microliters of flow buffer containing an antibody cocktail.

Resuspend the cells incubate at four degrees Celsius for 20 minutes. Then add an additional 150 microliters of flow buffer centrifuge at 500 times G for three minutes. Remove the SNAT and add 200 microliters of flow buffer before spinning again and discarding the supernatant with 100 microliters of 2%paraldehyde in PBS.

Re suspend the cells and incubate at four degrees Celsius for 30 minutes. Add 150 microliters of flow buffer and wash the cells as just demonstrated. Re suspend the cells in 200 microliters of flow buffer and transfer to flow tube inserts or standard flow tubes.

Add an additional 100 to 200 microliters of flow buffer and store at four degrees Celsius in the dark until carrying out flow cytometry for reliable flow cytometry results. Acquire at least 1000 donor cells per sample for samples from genetically modified or otherwise manipulated. Mice that show minimal expansion of donor cells collect an equivalent number of total lymphocytes as collection of so many donor cells may not be feasible as seen here.

Mice with SLE develops S splenomegaly in as little as 14 days exhibiting spleens two to three times the size of healthy mice in terms of mass and cellularity. Cytes are sequentially gated on unlike scatter elimination of doublets viable cells and CD four positive. TCR beta positive.

Donor cells are distinguished from recipient cells based on CD 45.1 and CD 45.2. Most donor cells differentiate into CXCR five PD one, double positive T follicular helper cells or TFHA portion of the recipient CD four T cell population also differentiates into TFH. The classification of TFH can be confirmed using additional markers including BCL six and ICOS.

This graph illustrates that after an initial die off and or migration of transferred cells, the expansion of donor derived TFH is logarithmic reaching 10 to 20 million cells in the spleens of mice 14 days after injection. CSFE labeling of transferred lymphocytes demonstrated that donor CD four T cells differentiate into TFH early after activation as all divided cells observed at days three, seven, and 14 upregulated CXCR five and PD one as shown here, the expansion of donor derived TFH is accompanied by a corresponding accumulation of endogenous plasma cells and GCB cells. Plasma cell accumulation in the spleen is delayed compared to that of TFH exhibiting no increase over naive animals on day three or seven.

Following this procedure, one can efficiently test the roles of candidate genes and the efficacy of novel therapies in a well-established model of SLE once mastered. This technique can be completed in two days if it is performed properly.