Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Our protocol enables the detailed identification and analysis of monocytes and macrophages and T-cells and their relevant subsets within the neurons and ovium and various other tissues of the immune system. Our technique facilitates the reliable harvest of life immune cells from the synovium and other relevant tissues for a comprehensive characterization of the osteoarthritic immune response. For the isolation of tissues of interest from an osteoarthritis model mouse, with the mouse in a supine position, wipe the chest, abdomen and legs with 70%ethanol.

Use scissors to carefully open the skin along the midline of the abdomen while leaving the abdominal cavity intact. And gently pull the skin on the right side of the animal away from the underlying muscle, leaving the subcutaneous adipose tissue attached to the skin. Place the mouse under a dissecting microscope and use two curved fine forceps to gently tease out the adipose tissue located at the thigh.

After identifying the three cross vessels, use fine dissecting forceps to remove the inguinal lymph node located at the intersection of the vessels, taking care of not to rupture the capsule. Use the forceps to remove the remaining fat from the surface of the lymph node and open the abdominal cavity. If no remaining fat can be seen, place the lymph node immediately in a previously prepared and labeled six well plate filled with 0.5 milliliters of RPMI media.

Hereafter, open the abdominal cavity. Use fine scissors to excise the spleen and to gently extract the intestines to expose the aorta and its bifurcation. Hereafter, place the spleen immediately in a previously prepared and labeled six well-played filled with 0.5 milliliters of RPMI media.

Remove the iliac lymph node located at the terminal segment of the abdominal aorta and the origin of the common iliac artery and carefully remove the skin from both hind limbs. Immediately place the lymph node in a previously prepared and labeled six well plate build with 0.5 milliliters of RPMI media pooling all lymph nodes from two animals. Using fine scissors or a scalpel clean the left femur of the attached muscle tissue and carefully disconnected both the stifle and the hip joint, leaving the whole bone intact while removing the femur.

Then placed the femur in a previously prepared and labeled six well plate filled with 0.5 milliliters of RPMI media. Identify the patella tendon of the right stifle joint and use fine scissors to remove the adjoining muscle tissue proximal to joint until approximately five milliliters of quadriceps tendon proximal to the patella are exposed. Cut through the quadriceps tendon approximately three to four millimeters proximal to the patella to form a handle and use fine forceps to gently pull the tension away from the joint to expose the edges of the joint capsule attachment.

Starting with the femur and moving toward the tibia, use a scalpel to carefully cut along the edges of the joint capsule on both sides while maintaining a gentle traction on the quadriceps tendon. When the synovial tissue block is attached only to the tibia, the intraarticular fat pad should be clearly visible distal to the patella and can be gently detached from the joint and anterior aspect of the Minsky. Then, cut along the remaining part of the joint capsule to remove the synovial tissue block and immediately place it in a previously prepared and labeled six well plate filled with 1.5 milliliters of RPMI media.

When all of the tissues have been collected, place two pooled spleens per condition onto a 70 micrometer cell strainer in a 15 milliliter tube and use a sterile three milliliter syringe plunger to gently mass rate the tissues through the mesh filter flushing the strainer frequently with a total of six milliliters of RPMI 1640 medium supplemented with 10%FBS. Sediment the cells by centrifugation, and re-suspend the pellet in five milliliters of red blood cell lysis puffer. After five minutes, stop the reaction with 10 milliliters of PBS and centrifuge the cells.

When no more red blood cells are observed, re-suspend the pellet in one milliliter of fresh PBS for counting. For lymph node processing place all four lymph nodes onto a 70 micrometer cell strainer in a new 15 milliliter tube and use a new sterile three milliliter plunger to gently tease the lymph nodes through the mesh into a single cell suspension, then centrifuge the cells at 500 times G for five minutes, discard the supernatant and re-suspend the pellet in 500 microliters of PBS for counting. To isolate the bone marrow, use a tissue thumb forceps to carefully grasp the intact femur and use sharp scissors to cut off the very end of the proximal femur.

Insert a 23 gauge needle into the middle of the intercondylar notch of the femur and rotate the needle while applying gentle pressure to drill a hole into the notch. Changing the needle as necessary, flush the bone contents with six milliliters of RPMI supplemented with 10%FBS onto a 70 micrometer cell strainer on a new 15 milliliter tube and use a new three milliliter syringe plunger to gently press the bone marrow through the mesh. Hereafter, perform the RBC step as demonstrated for the spleen.

When no more red blood cells are observed, re-suspend the pellet in one milliliter of fresh PBS for counting. For processing of the synovial tissue use fine surgical scissors to dice the two synovial tissue blocks into tiny pieces and transfer the samples into a new 15 milliliter tube. Rinse the collection container with 500 microliters of medium to collect any remaining cells and synovial tissues and pull the wash in the sample collection tube.

Next, add a sufficient volume of enzyme to achieve in a one unit per milliliter final concentration and digest the synovial tissue sample at 37 degrees Celsius in an incubator for two hours of a rotator. Ensure sufficient movement when placing the tubes in the rotator. At the end of the incubation, stop the digestion with eight milliliters of RPMI supplemented with 10%FBS and filter the cell suspension through a 70 micrometer cell strainer into a new 15 milliliter tube.

Rinse the old 15 milliliter tube with five milliliters of medium and use this to wash the filter. Then, sediment the cells by centrifugation and resuspend the pellet in 500 microliters of PBS for counting. To perform viability staining of the isolated immune cells, add five times 10 to the five cells of each cell suspension to the appropriate wells of two different 96 well U-bottom plates and collect the cells to the bottom of each well.

Always include sufficient unstained cells of each tissue type as negative controls. Wash cells with 200 microliters of PBS and re-suspend the pellets in 100 microliters of cell in permeant amine reactive dye per well for a 15 minute incubation at four degrees Celsius protected from light. At the end of the incubation wash the cells two times in 200 microliters of FACS buffer per wash and re-suspend each pellet in 100 microliters of the appropriate antibody of interest or control cocktail.

If both intra and extracellular staining is conducted in the same perform the extracellular staining step now. After 30 minute incubation at four degrees Celsius protected from light wash the cells two times with 200 microliters of FACS buffer per well and re-suspend the cells and the monocyte subset panel plate in 250 microliters of FACS buffer supplemented with one millimolar EDTA. Conduct this step only if no intracellular staining is planned, otherwise move on to the intracellular staining aspect of the protocol.

Then, transfer each monocyte subset sample into the appropriate corresponding FACS tube on ice protected from light. For intercellular staining of the T-cell subset panel cells, re-suspend the pellets in 200 microliters of fixation buffer from an appropriate fixation and permeabilization kit for a 40 minute incubation at four degrees Celsius protected from light. At the end of the incubation wash the cells two times with 200 microliters of permeabilization wash buffer and re-suspend the pellets in 100 microliters of the appropriate antibody of interest or control cocktail for a 40 minute incubation at four degrees Celsius protected from light.

Then, wash the cells two times in 200 microliters of permeabilization wash buffer per wash. And re-suspend the cells in 250 microliters of FACS plus EDTA buffer before transferring the samples into the appropriate corresponding FACS tubes on ice protected from light. Here, the hierarchical gating strategy for the monocyte subset panel on immune cells gathered from the bone marrow of DMM treated animals can be observed.

Unstained trolls can be used to determine the true negatives for the dead alive stain and the gates can be adjusted each time the experiment is conducted as necessary. In this representative analysis, immune cells were isolated from the synovial tissues and stained with extracellular surface markers, six weeks after DMM or sham control surgery. A higher percentage of Ly-SC positive MHC2 negative and M2 macrophages are observed in DMM treated animals compared to the cell populations observed in sham surgery mice.

Here, the hierarchical gating strategy for the extra and intracellular T-cell panel on immune cells isolated from the spleen of DMM treated animals can be observed. A higher percentage of TH1 cells are observed in the lymph nodes and synovial tissues of DMM animals as well as higher numbers of T regulatory cells and TH17 cells. To ensure the acquisition of high quality samples, the tissues must be harvested in a swift, meticulous and consistent manner.

Once the immune cells have been isolated, other downstream analysis can be conducted such as RTPCR, cell culture and rest on blood to shine a light on molecular processes of interest.