I work with Magnetic Activated Cell Sorting (MACS). I observed positive cells in negative fraction after positive selection. According to the flow cytometry performed after MACS, values of positive cells in fractions: unsorted, negative and positive were similar. What is more, I observed low yield after sorting. Usually I use 5-10 million of cells, having about 40-80% positive cells in population (differences depend on the donor and culture media we use) and usually receive after sorting not more than 200k positive cells. Last few times, the efficacy even lowered and I obtained not more than 50k cells after MACS.
How can I improve the yield of MACS Sorting? From my point of view, it seems like magnet columns do not select the whole positive population. How can I fix it? Do you observe similar situation after MACS?
Technical details: I work with AutoMACS Pro Separator, positive selection, beads: anti-SSEA-4, cells: Mesenchymal Stem Cells. I follow the protocol provided by producer of beads – together with added volume of beads, time and temperature of incubation. Only exception: separation is conducted on RT, with MACS buffer with RT – when I used cooled buffers, I observed huge mortality and almost none of cells survived.
We observed similar tendencies due to clogging of the cells when we used STRO-1 Antibodies and anti-IgM microbeads on our stem cells. One of the main techniqual steps that really improved our yield was passing the cells through strainers prior to passage through the columns to minimize clogging. This greatly improved our yield.
Karim Mohamed Fawzy El-Sayed, Rashmi Bhardwaj
Thank you! We use the strainers provided by Miltneyi (AutoMACS producer) to achieve single cells for separation but it doesn't change the yield in our case. Do you use strainers form Miltenyi or can you recommend other manufacturer?Rashmi Bhardwaj
I will try to increase the time of incubation. I considered to do this but AutoMACS representative discouraged me as it can decrease the specifity of beads (and decrease the purity).
Have you tried negative sorting prior to positive sorting?
When we used the Miltenyi system with mixed cultures of totipotent (CEA-CAM-1+), pluripotent (SSEA-4+), and mesodermal (Thy-1+) stem cells, we found that we could get greater yields of preferred cell type if we performed negative selections first with the opposing antibodies, then incubating the eluant with the preferred antibody.
For example, if we wanted to purify SSEA-4+ positive cells, we would first incubate the cell mixture with antibodies to CEA-CAM-1 and Thy-1 (same species of origin for both antibodies). Let those cells bind to the column and collect the eluant. Then incubate the eluant with antibody to SSEA-4+ (different species for primary antibody) and re-run eluant through second column. Also, the secondary antibodies used needed to be pre-absorbed to the species of the primary antibody. Meaning, if the primary antibody was of mouse origin, secondary antibody needed to be anything but mouse, such as rat (1st column). And the tertiary antibody (bound to the Miltenyi beads) needed to be anti-rat. For the second column, we would use a different secondary antibody (e.g., anti-hamster, anti-guinea pig, etc.). This would preclude any residual totipotent or mesodermal cells binding to the second column. And we could recover approximately 95-98% of the cells present.
To accomplish this we standardized our protocols with a mixture of known quantities of totipotent, pluripotent, and mesodermal stem cells in a mixed preparation, first. We tested primary antibodies, secondary antibodies, tertiary antibodies, incubation times, incubation temperatures, buffer solutions, column heights, column densities, buffer washes, etc. Once we could repeat the protocols with a >95% confidence, no matter who was performing the protocols (myself, my research associate, my research assistant, volunteers in the laboratory), we then moved on to experimentation.
Henry E Young That's really impresive work! To be honest, I considered applying negative selection. My collegue performed this program on other set of cells (CD146+ MSC) and saw no differences. However, her approach was way simpler - she just incubated cells with anit-SSEA-4 beads, then applied negative selection program and then treated negative fraction as positive.