QUESTIONS AND ANSWERS ABOUT CELL SORTING
Q 1: When should I use fluorescence activated cell sorting over bulk separation methods like panning or magnetic bead separations?
A: a) When very high purity (95%-100%) of the target population is required.
b) For separation of populations that have a low density of receptors on their surface.
c) For enrichment of populations on the basis of surface receptor density.
d) For separations on the basis of multicolor staining.
e) For separations on the basis of internal staining e.g of DNA or of internal antigens.
Q 2: Will my cells be harmed by the sorting process?
A: Generally, the cells will be not harmed through the process itself as long as they are maintained continually at a temperature, pH, and in media that is most suited to them.
Q 3: How many cells do I need to prepare to recover 1 X 106 of a population that comprises 10% of the cells?
A: 1 X 106 = 10 % target population x 50% recovery x 20 X 106 starting cell number. 50% recovery is a reasonable number, but the actual percentage of cells that are recovered with a particular sort depend on a multitude of factors:
a) Cell death that occurs pre- and post sort and loss through adherence of cells to tube walls.
b) Sort rate; the higher the sort rate the lower the recovery.
c) Precision of sort set up of the flow cytometer.
d) If it is an enrichment sort or a purity sort. Enrichment sorts have higher recovery than purity sorts.
Q 4: Are there ways to improve sort recovery?
A: Yes.
a) Use polypropylene tubes for cell processing and sorting.
b) Count cells immediately prior to sorting (after all washes).
c) Use blank Hank’s instead of PBS as sheath fluid.
d) Use polypropylene collection tubes and fill them > 1/3 with media that contains 20% serum.
e) Find optimal temperature for sort (4°C, 15°C, RT).
f) Invert tubes every hour during the sort.
g) Process collection tubes immediately after they are filled up or the sort is finished.
h) Spin collection tubes ~ 10 min.
Q 5: Do I have to use the monoclonal antibodies for staining of the surface antigens at the same cell to reagent ratio as for small cell numbers?
A: Our experience suggests that incubating 20 to 30 million cells in a volume of 0.5 ml of buffer with a quarter to a fifth the amount of antibody that one would calculate as the correct amount through scaling up is sufficient for adequate staining.
Q 6: How long does an average sort take?
A: The average sort takes about 1 hour of set up time for the instrument, up to 15 minutes for setting sort regions for the cells, and 30 minutes of post-sort analysis. Sorting of 20 X 106 cells takes about 2 to 4 hours. The actual time however, is highly dependent on the percentage of the desired cells in the sample as well as the purity and the number of cells that the investigator requested for the post sort assay. The set up time of the flow cytometer for a sterile sort is approximately 90 minutes.
A: a) When very high purity (95%-100%) of the target population is required.
b) For separation of populations that have a low density of receptors on their surface.
c) For enrichment of populations on the basis of surface receptor density.
d) For separations on the basis of multicolor staining.
e) For separations on the basis of internal staining e.g of DNA or of internal antigens.
Q 2: Will my cells be harmed by the sorting process?
A: Generally, the cells will be not harmed through the process itself as long as they are maintained continually at a temperature, pH, and in media that is most suited to them.
Q 3: How many cells do I need to prepare to recover 1 X 106 of a population that comprises 10% of the cells?
A: 1 X 106 = 10 % target population x 50% recovery x 20 X 106 starting cell number. 50% recovery is a reasonable number, but the actual percentage of cells that are recovered with a particular sort depend on a multitude of factors:
a) Cell death that occurs pre- and post sort and loss through adherence of cells to tube walls.
b) Sort rate; the higher the sort rate the lower the recovery.
c) Precision of sort set up of the flow cytometer.
d) If it is an enrichment sort or a purity sort. Enrichment sorts have higher recovery than purity sorts.
Q 4: Are there ways to improve sort recovery?
A: Yes.
a) Use polypropylene tubes for cell processing and sorting.
b) Count cells immediately prior to sorting (after all washes).
c) Use blank Hank’s instead of PBS as sheath fluid.
d) Use polypropylene collection tubes and fill them > 1/3 with media that contains 20% serum.
e) Find optimal temperature for sort (4°C, 15°C, RT).
f) Invert tubes every hour during the sort.
g) Process collection tubes immediately after they are filled up or the sort is finished.
h) Spin collection tubes ~ 10 min.
Q 5: Do I have to use the monoclonal antibodies for staining of the surface antigens at the same cell to reagent ratio as for small cell numbers?
A: Our experience suggests that incubating 20 to 30 million cells in a volume of 0.5 ml of buffer with a quarter to a fifth the amount of antibody that one would calculate as the correct amount through scaling up is sufficient for adequate staining.
Q 6: How long does an average sort take?
A: The average sort takes about 1 hour of set up time for the instrument, up to 15 minutes for setting sort regions for the cells, and 30 minutes of post-sort analysis. Sorting of 20 X 106 cells takes about 2 to 4 hours. The actual time however, is highly dependent on the percentage of the desired cells in the sample as well as the purity and the number of cells that the investigator requested for the post sort assay. The set up time of the flow cytometer for a sterile sort is approximately 90 minutes.
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