100 µg rituximab (Invivogen, anti-hCD20-hIgG1) was labelled using Alexa Fluor® 647 Monoclonal Antibody Labelling Kit (Invitrogen) following the protocol provided. After that, labelled rituximab was added to Raji cells (ATCC CCL-86) and Jurkat cells separately to test for CD20 specificity. Different concentrations of rituximab were tested to determine the minimum concentration required to saturate the cell surface with rituximab via comparison of geometrical means of different fluorescence intensities.
Raji cell culture
Raji cells were maintained in 90% RPMI media, 10% FCS (fetal calf serum) supplemented with 1% L-glutamine at 37°C, 5% CO2. Cells were split every 2–3 days to keep them at appropriate confluence.
Labelling of cells
15 µL of cells (around 7×106 cells/mL) were put in 75 µL cell-complete media (described above). 10 µL (0.1 mg/mL) of labelled rituximab was added onto the cells in an Eppendorf tube. This mix was then incubated for 1 h at 37°C, 5% CO2. Simultaneously, µ-Slide 8 well glass-bottom imaging chambers (Ibidi) were coated with 1 mg/mL BSA for 30 min and washed twice with 200 µL phenol red-free L15 medium, leaving 200 µL L15 in each well. Following the 1 h antibody incubation, 500 µL L15 was added to dilute unbound antibody. Cells were subsequently spun down (2000 rpm, 5 min, “Eppendorf mini-spin” centrifuge), supernatant was discarded, and the pelleted cells taken up in either (i) 300 µL PBS and transferred into the coated microscopy well (removing the L15 from the well) for imaging of labelled antibody only; or (ii) 100 µL L15 for other experiments. In case (ii), depending on the experiment, one or more of the following reagents were added: nuclear staining- 1 drop NucBlue® Live cell stain (Life Technologies) and incubation at RT for 5 min; mitochondrial staining- 0.5 µL (1 µM) MitoTracker® Orange CMTMRos (Thermofisher, #M7510) during antibody incubation and incubation for 30 min at 37°C. Thereafter, the mix was transferred into the imaging chamber containing 200 µL L15 media. Subsequent imaging was carried out on a Zeiss LSM 780 confocal microscope. Note: L15 serum-free media may be used to minimise cross-reactivity of antibody with serum. However, this comes at the cost of cell health and increased cell death. We hence recommend using cell media. Note: BSA coating prevents cell attachment to the well bottom. However, cell attachment in BSA-uncoated wells did not influence the number of polarised cells.
Actin disruption with Latrunculin B or Cytochalasin D
Before labelling, 1 µL (0.1 mM) (final concentration 1 µM) of Latrunculin B or Cytochalasin D was added to 75 µL complete-cell media and rituximab was subsequently added right away or after 30 min at 37°C, 5% CO2.
Visualization of PIP2
In order to visualise PIP2, the PH-PLCD1 domain from the PH-PLCD1-GFP plasmid (Addgene plasmid # 51407) was cloned into the standard lentiviral vector Phr. PH-PLCD1 domain integrity was verified by sequencing. Subsequently lentivirus was obtained by transfecting HEK293T cells with pQ8.91 0.5 µg, pMD-G 0.5 µg, pHR-PH-GFP 0.5 µg, Genejuice (Merck) 4.5 µL, DMEM (Sigma-Aldrich) 150 µL, milliQ water 20 µL. 48 h after transfection, supernatant was harvested and spun down to remove particulates. 0.5 mL of viral suspension was then added to 1.5 mL of 70% confluent Raji suspension. After 3 days, localisation of PIP2 was imaged after labeling with Rtx-Alexa647, as described previously, using the Zeiss LSM 780 confocal microscope.
Preparation of GPMVs
Raji cells were washed twice with 1 mL of hypotonic GPMV buffer (50 mM NaCl, 2 mM CaCl2, 10 mM HEPES; adjusted to pH 7.4 with HCl or NaOH). Then, 1 mL isotonic GPMV buffer (150 mM NaCl, 2 mM CaCl2, 10 mM HEPES; adjusted to pH 7.4 with HCl or NaOH) containing the vesiculation agent N-ethylmalemide (NEM, 2 mM final concentration) was added to cells. The cells were transferred to a 35 mm petri dish and incubated at 37°C for 1 h. Most of the dead cells attach to the bottom of the dish, making the isolation of floating GPMVs easy. The buffer containing GPMVs was gently transferred to an Eppendorf tube. To get rid of the remaining dead cells, the solution was spun down at 1000 rpm for 2 min in an “Eppendorf minispin” centrifuge. The GPMV-enriched supernatant was then used for further experiments, similar to cells in earlier described experiments.
Presented column statistics was done using GraphPad Prism, comparing measurements for two conditions through unpaired t-test analysis. Error bars in graphs represent standard deviations.