Name: Homing of Hematopoietic Cells to the Bone Marrow (Video) | JoVE Journal
Uploaded: 2009-03-18T00:00:00.000+00:00
Duration: 10 min 40 s
Description: Learn a protocol for studying hematopoietic cell homing to bone marrow niches using dye staining and tracking in recipient animals. This method enables comparison of homing efficiency between genetically distinct cell populations.

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5 minutes

This protocol was tested and optimized in the laboratory of Dr. David Scadden. Funding is provided by the Dana Farber Cancer Institute/Massachusetts General Hospital Clinical Hematology and Oncology Fellowship Program.

<ol>
<li> We begin the homing experiment by extracting cells to be used in the experiment. For this current experiment we are interested in finding out if the homing of whole bone marrow to the niches in the long bones of recipient animals is different for bone marrow cells extracted from WT animals such as C57BL/6J (WT) and those that are transgenic knockouts for Lysophosphatidic Receptor 1 (LPAR1) (KO).</li>
<li>Before we start the experiment, we will collect the materials we need for this experiment. In our lab all mouse dissections are carried out in a laminar flow tissue culture hood especially if the cells are going to be transplanted into a recipient animal instead of being used for ex-vivo analysis. We start by placing a blue sheet inside the hood. Other objects needed include a pair of forceps and a sharp pair of scissors which have been autoclaved , a sterile disposable scalpel and alcohol swabs. We will need modified PBS and a sterile 6 well plate, a mortar, pestle, 50cc blue topped conical tube and a 70uM disposable filter.</li>
<li>We start by taking one WT and one KO mouse.</li>
<li>These mice are euthanized by CO2 inhalation and then dipped in 70% isopropranolol for 5 seconds. This step prevents contamination of the cells of interest with mouse fur and in our hands has not led to any compromise in yield of cells or experimental results.</li>
<li>The mice are then dissected in turn using a pair of forceps and sharp scissors. We usually start with the WT mouse as a matter of routine. A small snip is made in the ventral skin of the mouse overlying the abdomen and the skin is then stretched using the hands. The skin comes off the hind limbs easily. The femur and tibia are then extracted taking care not to dislodge the head of the femur as this contains a large amount of bone marrow. The upper limbs are detached from the animal&rsquo;s torso by simply applying traction to them while keeping the torso immobile. This removes the upper limbs as one piece. The humeri are then extracted from the upper limbs.</li>
<li>The removed bones are placed in modified PBS which has been added to 6 well plates. Modified PBS is made by adding 2 ml of heat inactivated Fetal Bovine Serum and 2 cc of EDTA 0.5 M pH8. This solution is then filtered through a Nalgene 0.45 uM filter and can be stored at 4 degrees Celcius for at least a month.</li>
<li>It is essential to label the wells in the plate in order to be able to identify WT and KO bones correctly.</li>
<li>The mouse carcasses are removed and disposed off.</li>
<li>A new sheet is placed in the hood and the bones are cleaned using a disposable scalpel.</li>
<li>The cleaned bones are placed one mouse at a time in the mortar. Two ml of modified PBS is added to the mortar and the bones are crushed using a pestle. It is important to first fragment the bones with 10 to and fro movements of the pestle and then pulverize them with circular rotatory movements. I usually do 50 clockwise, then 50 counterclockwise movements and so on until the material left in the mortar is white in color and the modified PBS added to the material remains white or transparent.</li>
<li>After every set of 50 circular crushing movements, we transfer the fluid from the mortar to a 70 uM filter placed on top of a 50 ml conical tube. Do not forget to add 2 ml of PBS to the mortar after every transfer to the filter. You do not want to try and pulverize dry bones or leave them dry for any length of time as this will lead to apoptosis of cells.</li>
<li>Once the material in the mortar becomes white, the material in the filter is transferred to the mortar and the crushing is again performed. This ensures that the cells in the filter are not wasted. Usually it takes one or two sets of circular rotatory movements to again make the material in the mortar white in color at which time the extraction of cells is complete.</li>
<li>The solution in the conical tube is now centrifuged (please remember to keep the cells from the WT and KO mouse separate. Hence you will have two different tubes (one with WT cells, the other with KO cells). Centrifuge at 1200 rpm for 10 minutes.</li>
<li>Aspirate off the supernatant using separate aspirator pipettes for each tube. Add 2 cc of ACK lysis buffer to each pellet. This lysis buffer should be sterile. Incubate on ice for 5 minutes.</li>
<li>Add 8 cc of modified PBS to each tube. Take 10 ul from each tube and pipette into two separate wells in a 96 well plate. Place the tubes in the centrifuge and spin them at 1200 rpm for 10 minutes.</li>
<li>While the tubes are spinning, add 30 ul of tryphan blue dye and 60 ul of modified PBS to each well of the 96 well plate that carries the cells of interest.</li>
<li>Add 10 ul of this mixture (please mix well by pipetting up and down) to a hemocytometer and count the cells in the four outer squares.</li>
<li>The cell count in each sample per ml is the number of cells thus counted divided by 4 and multiplied by 100 000.</li>
<li>Once the centrifugation is complete, remove the tubes from the centrifuge and resuspend the cells in PBS without calcium, magnesium or L glutamine at a concentration of 20 million cells per ml. You will likely have about 5 ml of volume for the WT and 5 ml of volume for the KO animal. From this step onward we will be using PBS, not modified PBS for the protocol.</li>
<li>To each tube add DiI, a cell staining dye, to achieve a final concentration of 5uM. Vortex immediately for 10 seconds. Incubate at 37 degrees for 10 minutes and avoid exposure to light.</li>
<li>Fill the tubes with PBS and spin for 10 minutes at 1200 rpm.</li>
<li>After centrifugation, aspirate the supernatant and resuspend the cells in PBS at 40 million cells per ml.</li>
<li>Load 500 ul into 1 cc syringes with a 27G needle (5 syringes for the WT mouse and 5 syringes for the KO mice). It may be prudent to prepare 6 syringes for each cohort. Having one extra can help as tail vein injections which we are going to do next can be tricky.</li>
<li>Cover the syringes with foil in order to keep the DiI from disintegrating.</li>
<li>Inject 500 ul of the cell mixture into each recipient animal; (5 for each cohort).</li>
<li>These animals need to be irradiated with 9 Gy of irradiation 4-24 hours prior to injection.</li>
<li>48 hours after injection, harvest bone marrow from the hind limbs of each recipient animal.</li>
<li>Process as described above for the donors. Resuspend the cells from each animal in 1 cc of PBS. Obtain a cell count using a hemacytometer.</li>
<li>Read on the LSR II flow cytometer from BD or an equivalent instrument. The result can be expressed as the percentage or absolute number of cells in the analyzed bone marrow which are DiI positive.</li>
</ol>

<ol><li>Adams, G. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stem+cell+engraftment+at+the+endosteal+niche+is+specified+by+the+calcium-sensing+receptor%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Stem cell engraftment at the endosteal niche is specified by the calcium-sensing receptor.</a> Nature. 439 (7076), 599-603 (2006).</li>
<li>Broxmeyer, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemokines+in+hematopoiesis%5BTitle%5D)+AND+%22Curr+Opin+Hematol%22%5BJournal%5D)">Chemokines in hematopoiesis.</a> Curr Opin Hematol. 15 (1), 49-58 (2008).</li>
</ol>

Homing is the process whereby bone marrow cells including stem cells, progenitor cells and differentiated cells, their way into the bone marrow after being injected into the blood stream or bone marrow cavity of mice. As is obvious from the definition, a scientist may chose to study the homing of hematopoietic stem cells, progenitor cells or differentiated cells identified by immunophenotyping and isolated by cell sorting. Typically scientists inject bone marrow cells depleted for lineage markers and hence enriched for...

<table><tbody><tr><td>DPBS (Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Mediatech, Inc.</td><td>21-031-CV</td><td></td></tr><tr><td>Sterile Fetal Bovine Serum</td><td>Valley Biomedical, Inc.</td><td>BS3033</td><td></td></tr><tr><td>0.5M EDTA (pH 8.0)</td><td>Boston BioProducts</td><td>BM-150</td><td></td></tr><tr><td>Tryphan Blue solution</td><td>Mediatech, Inc.</td><td>25-900-CI</td><td></td></tr><tr><td>ACK Lysing Buffer</td><td>Lonza Inc.</td><td>10-548E</td><td></td></tr><tr><td>Isopropranolol U.S.P.</td><td>Denison Pharmaceuticals, Inc</td><td></td><td></td></tr><tr><td>Vybrant DiD cell-labelling solution</td><td>Invitrogen</td><td>V22887</td><td></td></tr><tr><td>Vybrant DiI cell-labelling solution</td><td>Invitrogen</td><td>V22885</td><td></td></tr></tbody></table>

homing of hematopoietic cells to the bone marrow

Homing of Hematopoietic Cells to the Bone Marrow

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow. Various chemomkines and receptors are involved in the homing of hematopoietic stem cells. [1, 2]
This paper outlines the classic homing protocol used in hematopoietic stem cell studies. In general this involves isolating the cell population whose homing needs to be investigated, staining this population with a dye of interest and injecting these cells into the blood stream of a recipient animal. The recipient animal is then sacrificed at a pre-determined time after injection and the bone marrow evaluated for the percentage or absolute number of cells which are positive for the dye of interest. In one of the most common experimental schemes, the homing efficiency of hematopoietic cells from two genetically distinct animals (a wild type animal and the corresponding knock-out) is compared. This article describes the hematopoietic cell homing protocol in the framework of such as experiment.

This article describes a protocol used to study the homing of hematopoietic cells to their niches in the bone marrow.

Homing is the phenomenon whereby transplanted hematopoietic cells are able to travel to and engraft or establish residence in the bone marrow.  Various ...

Homing is the phenomenon where transplanted hematopoietic cells find their way into the recipient's bone marrow. This article describes a protocol for studying the homing of hematopoietic cells to the relative areas in the bone marrow. By isolating the cell population of interest, staining this population with a dye and injecting them into the bloodstream of a recipient animal.The percentage of cells positive for the dye in the animal is then determined after a period of time. With this method, the homing efficiency of these cells from two genetically distinct animals can be compared. Hi, my name is RHI Reen Yusef, and I'm a postdoctoral fellow in Dr.David Skadden's laboratory at the Center for Regenerative Medicine at Massachusetts General Hospital and the Harvard Stem Cell Institute.Today we're going to be showing you a procedure to study the homing of hematopoietic stem cells to the bone marrow where they normally reside. We use this protocol in our laboratory to study the differences between the homing of hematopoietic stem cells from various genetically altered mice. So let's get started.To begin the homing experiment, we extract bone marrow from both WT and lysophosphatidic receptor one or LPA R one knockout animals. All mouse dissection should be done in a laminar flow tissue culture hood, especially if the cells are going to be transplanted into a recipient animal For dissection, first place a liner on the bottom of the hood. Next place dissection tools on the liner, including a pair of autoclave, forceps and sharp pair of scissors, a sterile disposable scalpel and alcohol swaps.Next place in the hood, a bottle of modified PBSA sterile six well plate a mortar and pestle, A 50 cc blue topped conical tube and a disposable filter. Following CO2 mediated euthanasia, sterilized the mouse's body in 70%isopropanol for five seconds to prevent contamination of the cells of interest. With mouse fur, the step does not seem to compromise the yield of cells or experimental results.After sterilizing the mouse, use a pair of forceps and sharp scissors to make a small snip in the ventral skin of the mouse over the abdomen. Next, using your hands stretch the skin off. The skin should come off the hind limbs.Easily extract the femur and tibia carefully to prevent dislodging the head of the femur. Since this contains a large amount of bone marrow. Next, detach the upper limbs from the animal's torso by simply applying traction to them while keeping the torso immobile.This removes the upper limbs as one piece. Extract the hum eye from these upper limbs. Place the collected bones in modified PBS in six well plates labeled for WT samples.Repeat this dissection for KO mice and collect in a six well plate with modified PBS this time labeled for KO samples. Once dissection is completed, dispose of the mouse carcasses and place a new liner in the hood. Then clean the bones using a disposable scalpel.Place the cleaned bones in the mortar one mouse at a time. Today we've started with the WT samples. Add two milliliters of modified PBS to the mortar and crush the bones using a pestle.First fragment the bones by moving the pestle back and forth 10 times. Next, pulverize the bones with circular rotary movements. You'll do a series of 50 clockwise, then 50 counterclockwise movements.After the first set of 50 circular movements, transfer the fluid from the mortar to a 70 micrometer filter placed on top of a 50 milliliter conical tube. Do not forget to add two milliliters of PBS to the mortar. After every transfer of the filter, this will prevent the bones drying and leading to apoptosis of the cells.Once the material in the mortar becomes white, transfer the material left in the filter back to the mortar and begin crushing again. This ensures that the cells in the filter are not wasted. Repeat a set of circular movements one or two times to make the material in the mortar whiten color, which indicates the extraction is complete.Repeat this procedure for samples from the ka o mouse. Don't worry. The rest of the protocol is much less labor intensive.To collect the cells centrifuge, the individual WT and KO sample solution in conical tubes at 1200 RPM for 10 minutes aspirate off the super natin using separate aspirator pipettes for each tube. Next, add two cc of sterile, a CK lysis buffer to each pellet and incubate on ice. For five minutes, add h CCC of modified PBS to each tube.Then take 10 microliter samples from each tube and pipette into two separate wells. In a 96 well plate clearly labeled WT and kko. Place the tubes in the centrifuge and spin them at 1200 RPM for 10 minutes.While the tubes are spinning, add 30 microliters of trian blue dye and 60 microliters of modified PBS to each well of the 96 well plate with the cells of interest. Mix the cells and die well by pipetting up and down. Remove the tubes from the centrifuge and resus.Suspend the cells in PBS without calcium, magnesium, or L-glutamine at a concentration of 20 million cells per milliliter. This often averages to be about five milliliters of volume each for WT and KO samples. To label the cells add to each tube the cell staining dye die eye to a final concentration of five micro molars.Vortex immediately for 10 seconds after the dye is well mixed with the samples. Incubated 37 degrees for 10 minutes and avoid exposure to light. Next, fill the tubes with regular PBS, not modified PBS and spin for 10 minutes.At 1200 RPM, continue to use this PBS rather than the modified PBS From this point on. After centrifugation, aspirate the super natin and resuspend the cells in PBS at 40 million cells per milliliter. Load 500 microliters into one cc syringes with a 27 gauge needle.Five syringes for the WT mice and five syringes for the K mice. Cover the syringes with foil in order to keep the dye eye from disintegrating. Inject 500 microliters of the cell mixture into each recipient animal, which has been irradiated with nine grays of irradiation four to 24 hours prior to injection.Having an extra syringe ready can help as tail injections can be challenging. Eight hours after injection harvest the bone marrow from the hind limbs of each recipient animal. Next process, the bone marrow is done previously with donor samples.Resus suspend the cells from each animal in one cc of PBS and count the cell population using a hemo cytometer. Finally, analyze the samples on the LSR two flow cytometer from BD or an equivalent instrument. We've just shown you how to perform a homing assay on hematopoietic stem cells.The number of cells that you choose to inject into the recipient mice, the dye that you use to stain these cells and the time at which the recipients have sacrificed are all variables that will need to be predetermined. Before you perform your experiment, your question and hypothesis will determine the choice of these variables. So that's it.Thank you for watching and good luck with your experiments.

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Research

JoVE Journal

Biology

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Learn a protocol for studying hematopoietic cell homing to bone marrow niches using dye staining and tracking in recipient animals. This method enables comparison of homing efficiency between genetically distinct cell populations.

Homing of Hematopoietic Cells to the Bone Marrow

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