I need to know how to isolate CD133 cells from bone marrow.
I will be using the isolation microbead kit from miltenyi biotec. There are a few challenges I have that I cannot seem to get a straight answer to.
For one thing, I do not have access to a haemocytometer, so counting the cells is impossible. I have asked for estimations on cell numbers per ml of mononuclear cells and they gave them to me.
I have about 50 milliliters of bone marrow. From that, I will be isolating the cd133 cells.
I am assuming I will be centrifuging the bone marrow to get the mononuclear cells, then isolating the cd133 cells from that right?
If so, I should have how much (in terms of volume) mononuclear cells? About 10 milliliters right?
I asked for an estimation on cd133 cell numbers from 50 ml's of bone marrow and they said about 1x10e7.
So my questions are,
How am I supposed to isolate cd133 cells from the 10ml's of bone marrow mononuclear cells with the little columns!?? I am going to have to reuse columns I'm sure, since there are only about 25 in a pack.
I just don't understand the process at all. I hope someone will see this and give me a explanation on what to do to isolate cd133 cells from 10ml's of mononuclear cells.
Just try to follow the column protocol. it's also up to how mch precentage of CD133 positive cells in your bone marrow. It's no problem you can re -use the column. I have isolated CD133 positive cells from cancer cells. You should be fine. Good luck.
Hi Ellie,
I work with CD133+ hematopoietic stem cells. So I'll try to help. But I have several questions to help me understand your goals and background. 1, is this for a class project or a thesis? 2. What is your hypothesis? Why did you pick this project? I would not have thought a community college would have the facilities to conduct this type of project. Who is advising you on this project and do they have experience working with CD133+ hematopoietic stem cells?
If you don't have access to a hemocytometer, you probably don't have access to a flow cytometer. If true, this project has big problems! First you need the hemocytometer to determine the number of cells you have. If you plan on using estimates, your wasting your time and money. You need an accurate cell number so you can determine the number of beads to use. Not enough beads you get a low cell yield, too many beads you get non-specific binding resulting in contamination with other cells types. The flow cytometer is used to determine the number of CD133+ cells in your population prior to cell isolation and after cell isolation to determine purity.
Dear Ellie,
I have been using miltenyi beads routinely for the isolation of CD34 and Cd133 cells
You easily isolate the CD 133 cells from 10ml bone marrow using just one column since each of these columns can be used for a maximum of 2x 108 cells i.e around 200million cells. From a 10 ml bone marrow you will obviously get lesser than this, so u can easily isolate you cell of interest by just using one column. Since you are going use this for the first time. I suggest you read and understand the instruction manual carefully before starting your expt.
Now to address ur issue abt cell count, one you get the monnunclear cells after density gradient mtd you can resuspend the cells in a min vol of 500 ul of buffer and pass thro' the coloumn, so here u can avoid the step of counting, but only worry is how wud u know the cell count after passing thro' the coloumn, you shd also be able to know the purity of the isolated cells by FACS, so at least if not before the isolation procedure u will definatelu need to chk the count and purity afte the isolation. This will tell u the recovery and the characterize the purified population.
I suggest before u start ensure that u have the counting of the cells in place along with the provision for the FACS. This will help you improving your confidence level abt the technique.
All the Best!!!, Do let me know if u need any further assistance.
I agree with the answers.
1) You can resuspend your MNCs in a small volume of buffer (less than 1mL) after separation and washes so that you will definitely have less than 10mL. You need them in a small volume for incubation with the beads.
Protocol : https://www.miltenyibiotec.com/~/media/Images/Products/Import/0001200/IM0001254.ashx
2) You have to count them to know the amount of beads you need and to be sure that you have less after purification (as you will lose the CD133- cells after washes from the column).
hemocytometer costs about $200 and you might be able to borrow one from any lab.
3) you have to measure the cd133+ purity (and viability) by flow cytometry after purification ( As it is your first time, I would personnaly compare before and after).
Good luck
Hemocytometers are normally pretty cheap if not very accurate. If money fails you can make your own with a microscope slide to which you use epoxy glue to attach shoulders on either side of a central space. Use thick coverslips (No 2s) for the shoulders. Leave about 1 cm wide gap in the centre which is where .the fluid sample will go The top piece of the cytometer is also a No. 2 coverslip which bridges between the shoulders but is not glued to them so you can take it off for cleaning and reuse of the hemocytometer.
You need to measure the dimensions of the space (counting chamber) between the slide and the coverslip and this is done by using vernier micrometers or calipers to measure the vertical gap but if you measured the thickness of the coverslips you will have a good idea. For the linear size of the counting area measure the breadth and width of the space betweenlders. Thus calculate the volume.
If the new hemocytometer is really clean and free from grease fill the space from a fine Pasteur pipette and count the number of cells in the space using a microscope, preferably a phase contrast microscope. Then use the cell count in the space volume to calculate the number of cells per ml. If there are too many cells for easy counting dilute the sample till counting is easy. This method with care should allow you to measure the cell concentration with an error of about plus or minus ten percent.
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