Journal: Bio-protocol

Article Title: Quantitative Microscopy for Cell–Surface and Cell–Cell Interactions in Immunology

doi: 10.21769/BioProtoc.5427

Figure Lengend Snippet: (A) Surface preparation of 8-well chambered slides: wells are sequentially coated with biotin-BSA, NeutrAvidin, and either anti-CD16 or HER2 protein (for experimental and control conditions). Protein incubations are performed on a see-saw rocking shaker. Each coating step is followed by DPBS rinses. Positive and negative controls are included to assess NK cell activity as well as nonspecific binding to the surface. After coating, wells are filled with RPMI supplemented medium and incubated for 15 min at 37 °C. (B) NK cell preparation and live-cell imaging: primary human NK cells are isolated and characterized by flow cytometry as described in Gonzàlez et al. [12]. Cells are pre-incubated with bispecific antibodies (bsAb) (concentration 0.14–14,000 pM) before seeding onto HER2. BsAb is not added to the control well with an anti-CD16-coated surface. Live-cell RICM imaging is performed immediately after cell addition at 37 °C, capturing time-lapse sequences from multiple positions over a 10-min period.

Article Snippet: MACSxpress Whole Blood NK Cell Isolation kit (Milteny Biotec, catalog number: 130-127-695) 12.

Techniques: Control, Activity Assay, Binding Assay, Incubation, Live Cell Imaging, Isolation, Flow Cytometry, Concentration Assay, Imaging

Journal: Bio-protocol

Article Title: Quantitative Microscopy for Cell–Surface and Cell–Cell Interactions in Immunology

doi: 10.21769/BioProtoc.5427

Figure Lengend Snippet: (A) NK cells purification: primary human NK cells are isolated from whole blood and characterized by flow cytometry as described in Part I, Section B and in González et al. [12]. (B) Effector cells preparation: NK cells are incubated with the CFSE cytoplasmic green fluorescent marker for 20 min at 37 °C, before adding them to the target cells. (C) Target cells preparation: the nuclei of live MCF7- WT and MCF7-HER2+ cells (cultured overnight in a multiwell slide) are stained with Hoechst 33342 for 15 min at 37 °C. (D) Culture media containing propidium iodide (PI) and the selected bispecific antibody (bsAb) is added to the multiwell with the fluorescent target cells; then, fluorescent NK cells are also added to the selected wells, and live epifluorescence imaging is performed at 37 °C, capturing time-lapse sequences from multiple positions.

Article Snippet: MACSxpress Whole Blood NK Cell Isolation kit (Milteny Biotec, catalog number: 130-127-695) 12.

Techniques: Purification, Isolation, Flow Cytometry, Incubation, Marker, Cell Culture, Staining, Imaging

Journal: Bio-protocol

Article Title: Quantitative Microscopy for Cell–Surface and Cell–Cell Interactions in Immunology

doi: 10.21769/BioProtoc.5427

Figure Lengend Snippet: The model-free background correction tab is shown, with typical parameters to perform a median background reconstruction over image tiles and normalize the images. The corrected image stack is shown on the right side using the stack viewer of Celldetective (normalized RICM image of spreading primary NK cells).

Article Snippet: MACSxpress Whole Blood NK Cell Isolation kit (Milteny Biotec, catalog number: 130-127-695) 12.

Techniques:

Journal: Bio-protocol

Article Title: Quantitative Microscopy for Cell–Surface and Cell–Cell Interactions in Immunology

doi: 10.21769/BioProtoc.5427

Figure Lengend Snippet: Here, the neighborhood is computed between primary NK cells in the cell–surface spreading assay that may be hovering or spread at any given time point.

Article Snippet: MACSxpress Whole Blood NK Cell Isolation kit (Milteny Biotec, catalog number: 130-127-695) 12.

Techniques:

Journal: Bio-protocol

Article Title: Quantitative Microscopy for Cell–Surface and Cell–Cell Interactions in Immunology

doi: 10.21769/BioProtoc.5427

Figure Lengend Snippet: Interacting primary NK cells in the cell–surface spreading assay are explored with a dedicated viewer. The reference cell is selected first (green circle on the right side), allowing the view of all neighbor cells at each time point as moving segments. Clicking on one of the segments allows selecting a cell pair and viewing on the left side the associated measurements. Here, the blue time series is the mean normalized–reflection interference contrast microscopy (RICM) intensity for the reference cell. The red time series is the same quantity as its neighbor, showing that this specific neighbor cell spreads a few frames after the reference cell.

Article Snippet: MACSxpress Whole Blood NK Cell Isolation kit (Milteny Biotec, catalog number: 130-127-695) 12.

Techniques: Microscopy