Miltenyi Biotec offers a well known, wide range of reliable systems to perform human or mouse cell separations. Their MACS® cell separation reagents include several types of MACS® MicroBeads that can be used for positive selection or depletion. We have been using the CD14 MicroBeads in our lab for isolation of monocytes from human peripheral blood that we receive as buffy coats from a blood bank. The CD14 MicroBeads are beads conjugated to monoclonal mouse anti-human CD14 antibodies. The CD14 antigen belongs to the LPS receptor complex and is strongly expressed on monocytes.
In general terms, the procedure can be described as follows: CD14+ cells are magnetically labelled with CD14 MicroBeads. Then the cell suspension is loaded on a column already placed in the magnetic separator. The magnetically labelled CD14+cells are retained on the column. Cells depleted of CD14 run through the column. After removal of the column from the magnetic field, the magnetically retained CD14+ cells can be eluted. The protocol includes several wash steps.
The separator is both a magnetic field and a support for the column. The magnetic field is available in different sizes to accommodate various isolation ranges and numbers of columns. The separators are relatively expensive, but you’ll need to buy it only once and use it in all your procedures. Eventually, however, you may want add new or different magnetic fields. In our case, Citomed (the Miltenyi representation in Portugal) has kindly lent us a magnetic field for an undetermined period of time.
Columns cannot be reused and are sold in 3 different sizes. In our lab we use LS columns which can be loaded with 2 x 109 total cells and retain 108 CD14 cells. The passage through the column is easy but takes a fair bit of time.
The beads are sold in a 2 ml suspension and are extremely expensive. We have always followed the labelling recommendations from Miltenyi, which suggest adding 20ul of beads per 107 total cells and it works well. Each kit can label 109 CD14 total cells and based on that, the company claims that MicroBeads allow 100 separations. However, we have found that 2 ml of MicroBeads is almost insufficient for processing a single blood bag from one blood bank donor. Nevertheless, the kit enabled us to purify CD14+ cells with higher purity (>95%) than any other isolation method, except for Flow Cell Sorting.
In order to guarantee the purity of the collected samples, the density gradient centrifugation should be done properly to avoid contaminating neutrophils. Neutrophils weakly express CD14 and are retained in the column. We have also observed that when samples were collected more than 8 hours before the labelling, the purity of the CD14 cells fraction decreased significantly (to 85%). To increase the purity of the magnetically labelled fraction, Miltenyi suggests a second column passage which, according to our experience, helps in the case of lymphocyte contamination, but it is useless in the case of neutrophils and also lowers the total CD14 cell fraction.
The effect of binding an antibody to CD14 in monocytes is supposedly minimal and does not trigger signal transduction since CD14 lacks a cytoplasmatic domain. However, in some monocyte cultures, we observed a significant decrease in CD14 within the first day of culture. As all the other monocyte parameters were maintained in those cases, we supposed the CD14 antigen was endocyted after MicroBead binding and we ignored any effect in cell signalling. Nevertheless, the process of differentiation of monocytes into dendritic cells was not affected.
The MACS® cell separation systems are efficient and reliable. Although laborious and with several steps, the sterility of the reagents is preserved and any first time user with some cell culture experience will deal quite well with the system. The overall system is quite expensive but the only method that might supplant such efficiency is Flow Cell Sorting, which is unaffordable for the majority of laboratories.
Paula Videira, Ph.D.
Medical Sciences Faculty