Enzyme free dissociation buffer

MDAMB-231 cells (ATCC) and PC-3 cells (ATCC) were grown in DMEM (high glucose) (Thermo Fisher Scientific) medium. COS-7 cells (UC Berkeley Cell Culture Core Facility) were grown in RPMI 1640 (Thermo Fisher Scientific) medium. The medium is supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% penicillin/streptomycin (Thermo Fisher Scientific). They are authenticated using STR profiling by ATCC, and they are tested to be free from mycoplasma contamination. Cells were detached by enzyme-free dissociation buffer (Thermo Fisher Scientific) and then allowed to interact with indicated substrates. Cells were transfected by Lipofectamine 2000 (Thermo Fisher Scientific). Grb2-tdEos, SOS-tdEos, and CAAX-tdEos plasmids are the same as previously published (Oh et al., 2012 (link)). F-tractin-EGFP is maintained in Zaidel-Bar’s lab. NCK-mEos3.2 and N-WASP-mEos3.2 are cloned in Mechanobiology Institute Core Facility. For immunostaining, cells were fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton-X, followed by standard immunostaining protocol. Primary antibodies include rabbit anti-EphA2 (CST, 6997), rabbit anti-pErk (CST, 9101), and rabbit anti-pY588-EphA2 (CST, 12677). The secondary antibody is goat anti-rabbit antibody conjugated with Alexa 488 fluorophores (Thermo Fisher Scientific).

Transfected A1207 and U87 cells were washed in phosphate-buffered saline (PBS) and harvested with an enzyme-free dissociation buffer (Thermo Fisher Scientific, Darmstadt, Germany). The cells were incubated with the indicated phycoerythrin (PE)-labeled antibodies for 30 min at room temperature. Then, the cells were washed in PBS and the mean fluorescence intensity (MFI) of 1,000 cells was analyzed by a FACSLyric flow cytometry (BD, Heidelberg, Germany). The non-expressing NG2 cell line HEK293 was used as negative control.

CD14⁺ cells were isolated from bulk PBMCs using species-specific magnetic microbead enrichment kits (Miltenyi Biotec) following the manufacturer’s instructions.
To differentiate CD14⁺ monocytes into dendritic cells, monocytes were re-suspended in RPMI-10 containing 20 ng/ml GM-CSF and 10 ng/ml IL-4 and incubated for 6 days at 37°C/5% CO₂ with a change of media every second day. On day 6 post-plating, the media was changed to DC maturation cocktail (RPMI-10 containing 10 ng/ml TNF-α, 10 ng/ml IL-1β, 15 ng/ml IL-6, 1 ug/ml prostaglandin E2, 20 ng/ml GM-CSF and 10 ng/ml IL-4) and incubated overnight at 37°C/5% CO₂. Non-adherent cells were washed with cold PBS and collected. Adherent cells were collected using enzyme-free Dissociation Buffer (Gibco) and incubated for 5–15 min at 37°C/5% CO₂. Cells were pelleted by centrifugation at 300 xg for 10 min at RT, re-suspended and combined prior to counting, plating and characterization by flow cytometry.
To differentiate CD14⁺ monocytes into macrophages, monocytes were re-suspended in macrophage culture medium (50% RPMI-10, 50% KPB-M15 conditioned medium and 10 ng/ml M-CSF). Cells were cultured at 37°C/5% CO₂ for 7 days with feeding on days 2, 4 and 6 by removal and replacement of the medium. Cells were harvested in cold PBS containing 2 mM EDTA.

Jurkat cells were pre-treated with anti-TfR or left untreated as described above. For the experiment, 96 wells were coated with ICAM-I-Fc (2 μg/ml, diluted in PBS, R&D Biosystems, # 720-IC-050) or VCAM-I-Fc (4 μg/ml, diluted in PBS, Biolegend, #553,706) for 1 h at 37 °C. To determine the exact number of cells added per well, one well was left uncoated to recover all cells after the experiment (input). Coated wells were washed twice carefully with PBS. Subsequently, 1 × 10⁵ cells were added per well in 100 μl and incubated for 15 min to equilibrate temperature and pH. Then, activating mAb cocktail (anti-CD3ε + anti-CD28, 1.5 μg/ml and 1 μg/ml final, respectively), or plain medium or MnCl₂ (1 mM final) was added into corresponding wells and incubated 30 min at 37 °C. Input samples were collected for counting directly after incubation. Next, remaining wells were carefully washed twice with medium and adherent cells were dissociated with enzyme-free dissociation buffer (Gibco, #13,151,014). Cells were then resuspended and counted using a LSR Fortessa flow cytometer (BD Biosciences).