Supplementary MaterialsData_Sheet_1. with different small interfering RNAs (siRNAs). This method does

Supplementary MaterialsData_Sheet_1. with different small interfering RNAs (siRNAs). This method does not require specialist facilities or specific training and does not induce cell toxicity or inflammatory activation. We demonstrate that this protocol successfully decreases the expression of two important genes associated with AD, the triggering receptor expressed in myeloid cells 2 (TREM2) and CD33, in main microglia cell cultures. methods using different microglia cells lines (e.g., BV2, CHME3), induced pluripotent stem cell (iPS) derived cells or rodent main microglia cell cultures. Cell lines are convenient because they do not require isolation and can be expanded indefinitely to provide high yields. However, during immortalization and repeated passaging, they may have acquired different features that are not present under physiological conditions in main microglia cells (Butovsky et al., 2014). Working with iPS cells is also a very useful tool due to their capabilities to be transformed into different cell types, including microglia cells. On the other hand, the process for growth and transformation of iPS cells into microglia cells is usually a laborious and complicated process, with several different protocols to follow in the literature (Muffat et al., 2016; Brownjohn et al., 2018). Hence, working with main microglia cell cultures is important. However, working with main microglia cell cultures presents challenges. One of the main limitations is the low yield produced from each animal and their limited survival time period in the absence of astrocytes. Also, main microglia cell cultures are hard cells to VE-821 enzyme inhibitor transfect, providing low efficiency of transfection and also are quite vulnerable to death when using traditional methods of transfection. One of the ways to solve this problem has been to generate different transgenic mouse lines, as in the case of triggering receptor expressed in myeloid cells 2 (TREM2; Turnbull et al., VE-821 enzyme inhibitor 2006; Cheng et al., 2018; Filipello et al., 2018). Main microglia cells are VE-821 enzyme inhibitor then isolated from these mice. However, this Rabbit polyclonal to ADO process is usually expensive and takes several months before you obtained the desired transgenic collection. An alternative to generation of transgenic mice has been the use of transduction systems to overexpress or silence the expression of different protein targets. In particular, the use of lentiviral vectors has proven to be effective for this purpose (Masuda et al., 2013). However, the whole process can VE-821 enzyme inhibitor be challenging and requires the use of specific material (like class II security hoods) and special training for different type of tasks such as design of the computer virus sequence, choosing the right bacterial strain to avoid genomic rearrangements while amplifying the viral vector, stability of your viral stock to freeze VE-821 enzyme inhibitor and thaw cycles, efficiency of transduction depending on the concentration of your virus (computer virus tittering), or the usage of different reagents (for instance polybrene or fibronectin) to decrease the repulsive charges of the computer virus with the cell membranes to increase the transduction efficiency. Here, we describe a simple method to knockdown the expression of different genes in main microglia by using small interfering RNA (siRNA) and the Magnetofection? theory patented by OZ Biosciences as a method of transfection. The Magnetofection? method allow us to associate nucleic acids (in this case siRNA), with specific magnetic nanoparticles (made of iron oxide which is usually fully degradable). The producing molecular complexes are then concentrated and transported into cells through an appropriate magnetic field. Therefore, the exploitation of a magnetic pressure exerted upon the siRNAs allows a very quick concentration of the entire applied siRNA dose on cells, so that 100% of the cells get in contact with a significant vector dose and promotes cellular uptake. The cellular uptake of the genetic material is usually accomplished by endocytosis and pinocytosis, two natural biological processes. Consequently, membrane architecture and structure remain intact in contrast to other physical transfection methods that damage, create hole or electroshock the cell membranes. To illustrate the use of this method in main microglia we have knocked down the expression of TREM2 and CD33, two important genes whose mutations are considered a risk factor to develop AD (Griciuc et al., 2013; Colonna and Wang, 2016). Materials and Methods Reagents LPS from serotype typhimurium (Sigma, catalog number L6511) was used for this study. Isolectin GS-IB4 from (Alexa Fluor? 568 conjugate) was purchased from Thermo Fisher (catalog number “type”:”entrez-nucleotide”,”attrs”:”text”:”I21412″,”term_id”:”1601766″I21412). Glial-Mag kit was purchased from OZ Biosciences (catalog number “type”:”entrez-protein”,”attrs”:”text”:”KGL00250″,”term_id”:”695954551″KGL00250). The protocol used is based on the manufacturers recommendation, which we have optimized for any 24-well plate format (Supplementary Physique S1). The different siRNAs (positivesiGLO and unfavorable controlsnon-targeting and siTREM2 and siCD33) were purchased from Dharmacon (Horizon) and their sequences and catalogs figures are provided in Table ?Table1.1. A complete list of.