Question: Why is my cell recovery low after using the Dead Cell Removal protocol (CG000093)?
Answer: The Dead Cell Removal Kit contains Annexin V MicroBeads and Binding Buffer for the magnetic labeling of cell debris, dead cells, and dying cells. Dead Cell Removal MicroBeads recognize a phosphatidylserine moiety in the plasma membrane of apoptotic and dead cells. During dead cell depletion, dead cells are labeled with magnetic Dead Cell Removal MicroBeads and passed through a separation column. The magnetically labeled dead cells are retained within the column. The unlabeled living cells run through; this cell fraction is thus depleted of dead cells.
Some cell loss is expected when using the Dead Cell removal protocol. It is recommended to start with a sufficient number of cells to account for this loss. Refer to the manufacturer's protocol for recommended cell loads for each column type.
Contributing factors to increased cell loss may be:
1. Cell clumping: Dead Cells leak their contents, making them sticky, which in turn causes cell clumping. The pore size of the column used in this protocol is 30um. Thus, an increase in cell clumping can clog the column, restricting the flow of living cells. Filtering the sample with a 30um filter may help break up large clumps if cell clumping is observed.
2. Presentation of phosphatidylserine due to sample preparation
a.Freezing/thawing cells: The freezing/thawing process results in the presentation of phosphatidylserine on the cell surface [1,2]. When this happens, living cells presenting this moiety can be bound by the Annexin V beads present in the kit and subsequently retained within the column.
Adding a cryoprotectant to your frozen cell suspension can help minimize cell stress caused by freezing which leads to the presentation of phosphatidylserine. We have two freeze-thaw protocols on our support site which can be used as a reference for safe freezing and thawing of cells :
- Fresh Frozen Human-Mouse Cell Line Mixtures for Single Cell RNA Sequencing
- Fresh Frozen Human Peripheral Blood Mononuclear Cells for Single Cell RNA Sequencing
b. RBC lysis: RBC lysis results in increased phosphatidylserine exposure in surviving cells . As a result, living cells may be recognized as dead by the dead cell removal microbeads. In such cases, Dead Cell Removal can be carried out prior to RBC lysis.
c. Stress of tissue dissociation: The stress of tissue dissociation may result in phosphatidylserine being presented on the cell surface in otherwise viable cells . The tissue dissociation protocol may need to be optimized to reduce stress on cells.
d. Presence of activated platelets: When working with cell samples containing platelets,e.g., blood samples, wash samples carefully at low centrifugation speed (200×g) in order to remove platelets. Dead Cell Removal MicroBeads bind to activated platelets. Activated platelets also bind to leukocytes, e.g., monocytes. In this case, viable cells bound to activated platelets would be retained in the magnetic field and reduce the recovery of living cells.
1. Johnson L, Coorey CP, Marks DC. The hemostatic activity of cryopreserved platelets is mediated by phosphatidylserine-expressing platelets and platelet microparticles. Transfusion. 2014; 54:1917–1926
2. Isachenko et al. Long-Time Cooling before Cryopreservation Decreased Translocation of Phosphatidylserine (Ptd-L-Ser) in Human Ovarian Tissue. PLoS One. 2015 Jun 17;10(6):e0129108
3. Tait, Smith and Wood (1999). Measurement of Phosphatidylserine Exposure in Leukocytes and Platelets by Whole-Blood Flow Cytometry with Annexin V. Blood Cells, Molecules, and Diseases 25(5): 271-278
4.Hwang et al. (2005). Single Cell Dissociation Methods for Flow Cytometric Cell Death Analysis of Hypoxia-Ischemia Injured Newborn Rat Pup Brain. Korean J Pediatr 48:545-550
Products: Single Cell Gene Expression, Single Immune Profiling, Single Cell ATAC, Fixed RNA Profiling Gene Expression