How to isolate machrophages from 96-well plate? I don,t want to use cell scraper and trypsin solution and I don,t have a rubber policeman for isolation of cells. so whats an easy and safe way to isolate.
Good morning,
you can try to incubate the cells with HBSS or PBS without Mg2+ and without Ca2+ (cold). Both ions are necessary for attachment. Adherent intestinal enterocytes detach from the plate if you incubate them with this puffer... Best regards...
Hello Obaid Hayat
You can use 5mM EDTA in cold PBS (without Ca2+ and Mg2+) and incubate for 15 mins followed by gently pipetting few times to ensure detachment. Wash the cells to remove the EDTA and resuspend the cells in cell culture medium.
Best.
Hello Obaid,
you could try PromoCell's Macrophage Detachment Solution, which is chemically defined and non-enzymatic. It was especially designed for the gentle release of adherent macrophages and guarantees the best possible cell viability, even after prolonged exposure times. Unlike enzyme-based solutions, the PromoCell Macrophage Detachment Solution does not alter cell surface proteins and neutralization is not necessary.
Here's the link to the product page:
https://promocell.com/product/macrophage-detachment-solution-xf/
If there are any questions, please feel free let me know.
Constanze Nossol Thank you so much, did you have any idea about how to remove the infected machrophages from the 96 well plate? If we use the PBS, so did the intracellular parasite will be disrupted ?
I can't share the answer of Amer Basile (2019 October 5th) with you, so I made a copy for you:
"...First, Mg2+ and Ca2+ DO NOT chelate the Trypsine enzyme nor do they inhibit the Trypsine activity, they improve it..
Bivalent ions are used by membrane adhesion proteins to create cell-cell and cell-substrate linking. These proteins have what we call "Bivalent ions interaction sites" which are regions rich in acidic (basophile) amino acids (like Glutamate and Aspartate). In presence of bivalent cations like Mg2+, Ca2+, Mn2+ and more (and sometimes trivalent cations like Fe3+) the membrane adhesion proteins on different cells are "cross-linked" through a static attraction (where the 2+ ion acts as a bridge) between the negatively charged amino acids) thus promoting cell adhesion. This is one of the reasons for which Polystyrene is used in cell-culture plastic-ware; the Polystyrene surface is negatively charged, making it possible for the cells to attach in the presence of bivalent or trivalent cations (cells are unable/less-able to attach to polypropylene for example). You'll also see that many of the new "Cell dissociation Buffers" include not only Trypsine, but also EDTA, Titriplex or DTPA. This is because these compounds are known to be good chelators (fixators) of polyvalent cations. Thus, when these molecules are present in the medium (or dissociation buffer/solution), they will "steal" the Ca2+, Mg2+ and Fe2+/3+ from the membrane adhesion molecules, rupturing the protein-protein interaction and promoting a better cell dissociation.
PBS buffers that are free from Ca2+ and Mg2+ work in a similar way. PBS buffers contains the phosphate ion (PO4(3-)) which in presence of polyvalent cations, forms insoluble complexes (calcium phosphate and magnesium phosphate). This process depletes the medium/solution from its polyvalent cations, breaking the electrostatic protein-protein cell-bridging, thus resulting in the dissociation of the cells..."
I have no experience with intracellular parasites, but in my opinion the detachment with PBS (wo Mg/Ca) should have no influence on the intracellular paracites. As Malcom Nobre mentioned you can additionally apply EDTA for detachment. Incubate the cells with the puffer (cold) and pipette gently up and down. Than check via microscope if the macrophage detached from the plate... If not, you can extend the incubation time. Do you have to use a 96-well-format? It is much more easier to practise this in a 24-well or 12-well format... We use this in a 12 well-format with MoDCs...Best regards...
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