Click it edu alexa fluor 488 flow cytometry assay

Brains were first incubated in 50% formamide/50% 2× SSC at 65°C for 1 h, then in acid, 2 m HCl, at 37°C for 15 min, and finally in 0.1 m boric acid, pH 8.5, for 5 min at RT. Following a 3 × 5 min TBS washes, the brains were incubated for 12 h overnight in a 500 μl solution of Click-iT reaction mixture from the Click-iT EdU AlexaFluor-488 Flow Cytometry Assay (Invitrogen) at RT, with vigorous agitation on a tissue rocker. Mouse anti-BrdU antibody directly conjugated to AlexaFluor-555 (Invitrogen, 1:200) and primary anti-HuC/D mouse IgG2B monoclonal 16AII (Invitrogen, 1:200) was added to the 500 μl of Click-iT reaction mixture and incubated in 4°C for 5 d. Brains were washed in TBS 2 × 30 min and incubated for 12 h overnight in secondary AlexaFluor-633 goat anti-mouse IgG2B (Invitrogen, 1:200) in TBS at RT. The brains were washed in TBS 2 × 12 h and stored in TBS at 4°C.

Fish were euthanized through submersion in ice water for 10 min until operculum movements ceased. Heads were fixed at 4 °C overnight in 4% paraformaldehyde/0.1 M phoshate buffered saline (PBS). Brains were dissected out, transferred for cryoprotection in 30% sucrose/0.1 M PBS, embedded in optimal cutting temperature (O.C.T.) compound and stored at −80 °C. Brains were cut on a cryostat (Microm HM505E, Walldorf, Germany) in 20 μm coronal sections and placed on slides stored at −80 °C until processing. The slides were washed in 0.1 M PBS and incubated in Click-iT^® reaction cocktail for 30 min from the Click-iT^® EdU Alexa Fluor^® 488 Flow Cytometry Assay (Invitrogen, Carlsbad, CA USA). After rinsing in PBS, slides were mounted using Vectashield mounting medium (Vector, Burlingame, CA, USA).

Cell proliferation was measured using the Click-it EdU Alexa Fluor 488 Flow Cytometry Assay (Invitrogen). Briefly, cells (2 × 10⁵ per well) were incubated overnight on six-well plates, incubated with EdU for 2 h, and then stimulated for 1 h with EGF (50 ng/mL). Proliferation was assessed by flow cytometry on a FACS Calibur instrument (BD Biosciences), and the data were analyzed using the Cell Quest software (BD Biosciences). Each experiment was repeated at least three times. Expression of cell surface EGFR was monitored by flow cytometry on living cells without permeabilization. After washing in ice-cold PBS, cells were immunolabeled on ice for 10 min with APC-conjugated anti-EGFR (Biolegend, #BLE352906, 1/100, Ozyme) in PBS containing 2% BSA. After two further washes in ice-cold PBS containing 2% BSA and one wash in ice-cold PBS, cells were suspended in PBS and analyzed by flow cytometry on a FACS Calibur instrument; data were analyzed using the Cell Quest software. Measurements were compared to the isotopic control (APC-conjugated anti-mouse IgG1, Biolegend clone MOPC-21 #BLE400122, 1/100, Ozyme) to determine background and positivity thresholds. Each experiment was repeated at least three times.

OVCAR5 were seeded in 12-well plates at 5300 cells/cm², allowed to grow for 2 days, and then serum-starved for 24 h (resulting in a final density of 126,000 cells/well). IGF2 was then either spiked directly into conditioned media (which contains cell-secreted IGFBPs, [27 (link)]) or serum-free media was aspirated, cells were rinsed once with PBS, and the IGF2 treatment was added with fresh serum-free media. For experiments with IGFBP3, 15 nM of recombinant human IGFBP3 (Peprotech) was added 30 min prior to treatment with IGF2. All experiments were done with 1 mL of media per well. Cell proliferation was quantified after 24 h of IGF2 treatment using the Click-iT® EdU Alexa Fluor® 488 flow cytometry assay (Life Technologies) according to manufacturer’s instructions. Cells were incubated with EdU for 6 h prior to sample collection and analyzed on an Accuri C6 flow cytometer (BD, Franklin Lakes, NJ). To investigate the effects of IGF2R knockdown and overexpression on IGF2-induced proliferation, transfected cells were plated as described and treated with either vehicle or 1 nM IGF2.

HGSOC cells were plated in 12-well plates at the same cell densities as the MMP2 expression assay and allowed to attach overnight. Cells were then rinsed with PBS and serum-free media was added. After serum starving for 24 h, cells were washed with PBS and stimulated with vehicle (0.1 % BSA in PBS) or 10 ng/mL HB-EGF, NRG1β, IGF1, or HGF in serum-free media. After 24 h of treatment, cell proliferation was quantified using the Click-iT^® EdU Alexa Fluor^® 488 flow cytometry assay (Life Technologies) according to manufacturer’s instructions. Cells were incubated with EdU for 6 h prior to sample collection and analyzed on a BD Accuri™ C6 flow cytometer (BD; Franklin Lakes, NJ, USA). Samples were gated for the EdU-positive population to determine the percentage of cells that entered S-phase during the EdU incubation.

Before staining, BMMNCs were treated with EasyLyse Erythrocyte Lysing Reagent (DAKO, Glostrup, Denmark, www.dako.com). For fluorescence‐activated cell sorting (FACS) analysis, 10⁶ to 10⁷ cells were stained and all cells gated as positive showed a fluorescence intensity greater than that detected on 99% of the cells labeled with the isotype‐matched control. Nonspecific antibody binding was blocked with phosphate‐buffered saline (PBS) containing 1% bovine serum albumin (BSA), 10% human serum, and 3% sera from species in which the secondary antibodies were raised. Staining was performed at 4°C for 30 minutes, before washing (with 2 mM EDTA, 0.5% BSA in PBS). Antibodies used are listed in Supporting Information Table 1. For analysis of proliferation, a Click‐iT EdU Alexa Fluor 488 Flow Cytometry Assay was used (Life Technologies, Carlsbad, CA, www.thermofisher.com) and for apoptosis/necrosis detection, Annexin V Apoptosis Detection Kit eFluor 450 (eBioscience, San Diego, CA, www.ebioscience.com). Samples were assayed on a FACS Calibur/Canto II cytometer (BD Biosciences, San Jose, CA, www.bdbiosciences.com) and analyzed using FlowJo v10 software (Ashland, OR, www.flowjo.com).