12/24/2022 0 Comments Flowjo 7.6.5 free download![]() ![]() Reagents used were: Dulbecco's Modified Eagle Medium (DMEM) (Gibco cat no. Our protocol has been validated on six adherent cell lines, representing breast, cervical, prostate, and fibrosarcoma cancer types and normal fibroblasts, using four commercially available dyes and two antibodies results have been obtained and confirmed utilizing two cytometry machines: a high-throughput flow cytometer (Accuri C6 HTFC Screening System) and the BD LSRFortessa.Īll cell lines used were obtained from ATCC (Manassas, VA): HeLa (human cervical adenocarcinoma ATCC CCL-2) MCF-7 (human breast adenocarcinoma ATCC HTB-22) BT20 (human breast carcinoma, ATCC HTB-19) DU145 (human prostate carcinoma ATCC HTB-81) HT1080 (human fibrosarcoma ATCC CCL-121) and BJ (human skin fibroblasts ATCC CRL-2522). The benefits of using 2.9 mM EDTA (pH 6.14) as described in our protocol (versus conventionally used concentrations of trypsin or trypsin/EDTA) are: no enzymatic inactivation is required, cell surface markers are conserved, cell viability is extended (up to 3 h), and the protocol is relatively simple. However, non-enzymatic methods (such as EDTA) have been recommended over enzymatic methods to detach adherent cells for FC ( 12) and for passaging and colony expansion of human pluripotent stem cells ( 13). Conventionally, 0.25% or 0.05% trypsin, both in an 0.53 mM EDTA (a chelator of divalent and trivalent ions) solution, is used for enzymatic detachment of adherent cells ( 12). (B) Comparison of key attributes of our protocol and the conventional protocol.Īdherent cells express protein molecules and receptors, including E-cadherins ( 7, 8) and integrins, which are involved in cell-to-cell adhesion and extracellular matrix attachments ( 9) and require Ca 2+, Mg 2+, and Mn 2+ for proper function ( 10, 11). ![]() Our two-step cell detaching protocol simply involves adding EDTA to cells in culture medium, waiting until the cells detach, adding stain to the medium and then subjecting cells to FC. (A) The conventional protocol involves multiple steps, including removing medium, adding trypsin/EDTA, transferring cells to tubes for centrifugation, washing cell pellets with PBS, staining and washing again by centrifugation, re-suspending cells, and then transferring cells to microplates for FC. ![]() Comparison of our protocol for preparing adherent cells for flow cytometry (FC) with the conventional protocol. The advantages of our protocol over the conventional protocol ( 5, 6) are listed in Figure 1B. To bypass these problems, we present a protocol using EDTA as a cell detachment reagent that allows direct culture, detachment, and staining of cells (with membrane-permeable dyes and surface antibodies) on microplates. Classically, adherent cells are detached with trypsin/EDTA and subjected to various wash and centrifugation steps before staining and transfer to microplates for FC ( Figure 1A). Because suspending adherent cells is laborious and time-consuming ( 2), the applicability of FC to high-throughput screening (HS) in 96- and 384- (or more) well plates is limited ( 3, 4). Although FC provides the most accurate quantitative measure of labeled cells in diagnostics, research, and drug discovery applications, its use is limited to cells in suspension ( 1). Since its inception and commercialization in the 1960s, flow cytometry (FC) has emerged as an indispensible tool for cell biology research. ![]()
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