Preparation of Whole Bone Marrow for Mass Cytometry Analysis of Neutrophil-lineage Cells

This mass cytometry protocol preserves the integrity of the whole bone marrow, allowing rescue of the information that was lost during conventional sample preparation for analysis of under studied cells. This fast and effortless bone marrow isolation protocol facilitates the acquisition of viable short-lived myeloid cell populations such as rare neutrophil-lineage subsets. Using mass cytometry to identify previously under appreciated immune cells such as neutrophil-lineage cells may have broad implications for a wide variety of diseases.

This protocol preserves the integrity of the short lived myeloid cells that are present in the bone marrow and can easily be applied to other tissue types such as the solid tumor. We simplified to protocol to make it as user friendly as possible, but as in all biological studies it may need a few practice runs to manage. When you’re doing this the first time, you can get all your reagents ready first and then strictly follow the protocol.

If you do experience any issues, you can connect to the CyTOForum and talk to other CyTOF users who can help troubleshoot. For biological sample preparation, simple tricks can make a huge difference with the end result. A visual demonstration is much easier for a new user to grasp the protocol.

If you are doing this for the first time, you can get your reagents ready first and then strictly follow the protocol. For bone marrow harvest, place a six to 10 week old C57 black six mouse in the supine position on a sterile surgical pad and use 70%ethanol to sterilize the abdomen and hind limbs. Use a pair of dissecting surgical scissors to open the abdominal cavity and remove the skin to expose the hind limbs.

Using a pair of blunt tipped dressing forceps, hold the mouse tibia right below the ankle and use a pair of curved dressing forceps to stabilize the tibia below the blunt tipped dressing forceps. Use the blunt tipped dressing forceps to break the tibia and remove the muscle to expose the bone. Before placing the tibia in cold PBS, move the stabilizing curved dressing forceps to the femur and slide the blunt tipped dressing forceps below the knee joint to hold the kneecap.

Gently pull up to dislocate the kneecap and remove the muscle from the kneecap to expose the femur. Hold the exposed femur by the curved dressing forceps and use surgical scissors to cut the femur from the bottom of the bone. Then place the femur in PBS.

Next, use an 18 gauge needle to punch a hole in a 0.5 milliliter micro centrifuge tube and place both bones in the tube with the open ends of the bones facing downward into the hole. Place the 0.5 milliliter tube into a 1.7 milliliter micro centrifuge tube and spin the double layered tubes in a micro centrifuge. At the end of the centrifugation, confirm that the bone marrow has been extracted to the bottom of the tube.

To stain the bone marrow cells for mass cytometry, re suspend the bone marrow in one milliliter of red blood cell lysis buffer for 10 minutes at room temperature. At the end of the incubation, collect the cells by centrifugation and re suspend the pellet in one milliliter of cold PBS. Filter the cells through a 70 micrometer strainer into a 15 milliliter conical tube and wash the cells with nine milliliters of fresh, cold PBS.

Re suspend the bone marrow cell pellet in 10 milliliters of fresh, cold PBS for counting and transfer a five times 10 to the sixth cell aliquot into a new 15 milliliter tube. Collect the cells by centrifugation and re suspend the cells in one milliliter of mass cytometry staining buffer, supplemented with 125 nanomolar cisplatin. After five minutes at room temperature, wash the cells in four milliliters of fresh mass cytometry buffer.

Re suspend the pellet in 50 microliters of FC receptor blocking solution. After 10 minutes at four degrees Celsius, add 50 microliters of the antibody cocktail of interest to the cells and gently pipet to mix. The temperature in different incubation steps are very important in preserving the viability of the bone marrow cells.

After 30 minutes at four degrees Celsius, wash the cells two times in two milliliters of fresh mass cytometry buffer per wash before re suspending the cells in one milliliter of freshly prepared 1.6%formaldehyde for 15 minutes at room temperature. At the end of the incubation, collect the cells by centrifugation and re suspend the pellet in one milliliter of fixed perm buffer supplemented with 125 nanomolar intercalation solution for an overnight incubation at four degrees Celsius. Before analyzing the cells by mass cytometry, gently vortex and centrifuge the cell suspension before washing the cells in two milliliters of fresh mass cytometry staining buffer.

Next, wash the cells two times in one milliliter of distilled water per wash, carefully aspirating the supernatant after the second wash before re suspending the cells at a one times 10 to the sixth cells per milliliter of mass cytometry buffer concentration for mass cytometry analysis. In this T distributed stochastic neighbor embedding plot, the cells across multiple mouse tissues were clustered into subsets based on the similarity of their surface marker expression profiles as measured by a 33 parameter mass cytometry panel. Cells with more similar properties were automatically clustered together based on the expression of these markers on each cell.

Using this method, the integrity of the whole bone marrow was preserved, leading to the discovery of a previously unknown cell population that simultaneously co-expresses neutrophil and hematopoietic stem and progenitor cells signature surface markers. This cell population, which was previously omitted in myeloid progenitor studies, due to Ly6G positive cell depletion, exhibits a distinct pattern of surface marker expression. More importantly, these data led to the discovery of a small subset of the Ly6G positive cell cluster that doesn’t express Ly6G, but was clustered closely to the CD117 positive Ly6G positive cells, suggesting the similarity of these cells to the neutrophil-lineage based on the expression of the 33 surface markers used in this mass cytometry experiment.

A 13 color florescence activated cell sorting panel could then be built, allowing the isolation of the neutrophil progenitors by flow cytometry for downstream functional assays. It is important to perform the bone marrow isolation procedure as quickly as possible and keep the cells at four degrees Celsius during the staining procedure.