TY - JOUR
T1 - Flow cytometry conjugate formation assay between natural killer cells and their target cells
AU - Iserentant, Gilles
AU - Seguin-Devaux, Carole
AU - Zimmer, Jacques
N1 - Publisher Copyright:
© 2024 Elsevier Inc.
PY - 2024/3/11
Y1 - 2024/3/11
N2 - Before being able to kill other cells, natural killer (NK) cells first have to establish contact with those targets. In case of a predominance of activating signals from the target cell over inhibitory ones, the killing process is initiated. It is possible, with a simple two-color flow cytometry method, to evaluate, for any given effector cell–target cell pair, the number of conjugates between both types of cells. The percentage obtained gives an idea of the amplitude of binding of the NK cells to the targets and might be expected to be indicative of the level of cytotoxicity. Nevertheless, there is no absolute correlation, as the percentages of conjugates are sometimes higher with relatively resistant targets than with the highly sensitive cell line K562. Practically, NK cells and target cells are stained with two differently fluorescent dyes and incubated together at the desired effector:target ratio (in our example, 1:1) for various periods of time (0, 10, 30 min, etc.) at 37 °C. After the incubation time, the cells are carefully introduced into the flow cytometer, where in principle three populations are distinguished: the single positive, unconjugated effector and target cells, respectively, and the double positive subset, which corresponds to the conjugates between both cell types. We describe here in detail the staining and cell culture protocols and procedures, and give several examples. Thus, the very cytotoxic NK leukemia cell line KHYG-1 versus the myeloid leukemia K562 (the “conventional” NK cell target) and the Burkitt lymphoma cell line Raji forms a high number of conjugates. In contrast, purified, non-activated, healthy donor-derived peripheral blood NK cells bind less to the targets, in accordance with their low (K562) or absent (Raji) cytotoxic activity.
AB - Before being able to kill other cells, natural killer (NK) cells first have to establish contact with those targets. In case of a predominance of activating signals from the target cell over inhibitory ones, the killing process is initiated. It is possible, with a simple two-color flow cytometry method, to evaluate, for any given effector cell–target cell pair, the number of conjugates between both types of cells. The percentage obtained gives an idea of the amplitude of binding of the NK cells to the targets and might be expected to be indicative of the level of cytotoxicity. Nevertheless, there is no absolute correlation, as the percentages of conjugates are sometimes higher with relatively resistant targets than with the highly sensitive cell line K562. Practically, NK cells and target cells are stained with two differently fluorescent dyes and incubated together at the desired effector:target ratio (in our example, 1:1) for various periods of time (0, 10, 30 min, etc.) at 37 °C. After the incubation time, the cells are carefully introduced into the flow cytometer, where in principle three populations are distinguished: the single positive, unconjugated effector and target cells, respectively, and the double positive subset, which corresponds to the conjugates between both cell types. We describe here in detail the staining and cell culture protocols and procedures, and give several examples. Thus, the very cytotoxic NK leukemia cell line KHYG-1 versus the myeloid leukemia K562 (the “conventional” NK cell target) and the Burkitt lymphoma cell line Raji forms a high number of conjugates. In contrast, purified, non-activated, healthy donor-derived peripheral blood NK cells bind less to the targets, in accordance with their low (K562) or absent (Raji) cytotoxic activity.
KW - Conjugate formation assay
KW - Flow cytometry
KW - Natural killer cells
KW - NK cells
KW - Target cells
UR - http://www.scopus.com/inward/record.url?scp=85187560366&partnerID=8YFLogxK
U2 - 10.1016/bs.mcb.2024.02.037
DO - 10.1016/bs.mcb.2024.02.037
M3 - Article
AN - SCOPUS:85187560366
SN - 0091-679X
JO - Methods in Cell Biology
JF - Methods in Cell Biology
ER -