Mem 258

Manufactured by Abcam

Sourced in United Kingdom

MEM-258 is a monoclonal antibody that recognizes the mouse CD258 antigen. CD258 is also known as LIGHT, which is a member of the tumor necrosis factor (TNF) superfamily. The antibody can be used for the identification and characterization of CD258-positive cells by various immunological methods.

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Lab products found in correlation

Accuri c6, by BD (4 mentions) Alexa fluor 647 goat anti mouse antibody, by Thermo Fisher Scientific (2 mentions) Nitrocellulose membrane, by GE Healthcare (1 mentions) Phospho stat3 ser727, by Cell Signaling Technology (1 mentions) β tubulin, by Merck Group (1 mentions) Mouse igg control antibody, by Santa Cruz Biotechnology (1 mentions)

4 protocols using mem 258

Profiling Receptor Expression in EOC Cells

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Receptor expression was profiled essentially as previously described.^{15 (link)} In brief, EOC cells (1.5 × 10⁵ cells per well in a 96-well plate) were seeded and washed in 200 μl of wash buffer (phosphate-buffered saline/1% bovine serum albumin) and incubated with 100 μl of wash buffer containing 1:500 of mouse anti-human monoclonal antibody against CAR (RmcB, Millipore, Watford, UK), 1:500 of mouse anti-human CD46 (MEM-258, Abcam, Cambridge, UK) or mouse immunoglobulin G control antibody (Santa Cruz Biotechnology, Heidelberg, Germany) for 1 h on ice. Cells were washed three times and incubated with a 1:500 dilution of goat anti-mouse Alexa Fluor 647 antibody (Invitrogen, Paisley, UK) for 1 h on ice. Cells were fixed in 4% paraformaldehyde for a minimum of 10 min at 4 °C for flow cytometry. In all, 2 × 10⁴ gated events were acquired in channel FL-4 on a BD Accuri C6 (BD Biosciences, San Jose, CA, USA) flow cytometer and data were analyzed in the BD Accuri C6 software version 1.0.264.21 (Becton Dickinson, Franklin Lakes, NJ, USA).

Hulin-Curtis S.L., Uusi-Kerttula H., Jones R., Hanna L., Chester J.D, & Parker A.L. (2016). Evaluation of CD46 re-targeted adenoviral vectors for clinical ovarian cancer intraperitoneal therapy. Cancer Gene Therapy, 23(7), 229-234.

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Monoclonal Antibodies for Cell Surface Markers

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Purified GB24 (mouse monoclonal raised against human CD46)was provided by Prof John Atkinson/Paula Bertram (Washington University School of Medicine, St. Louis, MO, USA). Purified MBC-1 (mouse monoclonal raised against CD55) was a gift from Prof Claire Harris (Cardiff University School of Medicine, Cardiff, UK). MEM-258 (mouse anti-Human CD46) was obtained from Abcam (Cambridge, UK). Hybridoma producing the mouse monoclonal antibody 543 was obtained from EACAC and purified from cell supernatant in house using protein G affinity column and standard protocols. Culture supernatant containing BRIC229 (mouse monoclonal raised against CD59) and anti-serum with reactivity to the Knops blood group antigen (Kn^a) was obtained from the International blood group reference laboratories (NHS BTS, Bristol, UK). Goat or donkey anti-Human IgG specific – [horse radishperoxidase] HRPO; (#109-035-008), phycoerythrin conjugated(#705-116-147) and sheep anti-mouse-IgG-HRPO (#515-035-071)were obtained from Stratech Scientific, Ltd. (Newmarket, UK).

Watson R., Wearmouth E., McLoughlin A.C., Jackson A., Ward S., Bertram P., Bennaceur K., Barker C.E., Pappworth I.Y., Kavanagh D., Lea S.M., Atkinson J.P., Goodship T.H, & Marchbank K.J. (2014). Autoantibodies to CD59, CD55, CD46 or CD35 are not associated with atypical haemolytic uraemic syndrome (aHUS). Molecular immunology, 63(2), 287-296.

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Western Blot Analysis of Immune Proteins

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Cells were lysed in RIPA buffer containing 10 mM Tris-HCl at pH 8.0, 1 mM EDTA, 140 mM NaCl, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100 and a protease inhibitor cocktail (AMRESCO LLC, Solon, OH). The cell suspension was incubated at 4°C for 15 min and then centrifuged at 14,000 g for 15 min at 4°C. The supernatant was collected and the protein concentration was measured by BCA assay (Pierce, Rockford, IL). The protein was separated by electrophoresis on SDS-PAGE and transferred onto a nitrocellulose membrane (GE Healthcare, Piscataway, NJ). Immunodetection was performed with the mouse monoclonal antibodies to CD46 (MEM-258, Abcam) and β-tubulin (7B9, Sigma, St. Louis, MO), rabbit polyclonal antibodies to STAT3 (#9132, Cell Signaling Technology, Danvers, MA) and phospho-STAT3 Ser727 (9134, Cell Signaling Technology), and a rabbit monoclonal antibody to phopho-STAT3 Tyr705 (D3A7, Cell Signaling Technology) in addition to the antibodies to CD55 and CD59, all used at 1∶1000 dilution.

Lee M.S., Jones T., Song D.Y., Jang J.H., Jung J.U, & Gao S.J. (2014). Exploitation of the Complement System by Oncogenic Kaposi's Sarcoma-Associated Herpesvirus for Cell Survival and Persistent Infection. PLoS Pathogens, 10(9), e1004412.

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Cell Surface Receptor Expression Profiling

Cited 1 time

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Receptor expression was profiled essentially as previously described.16 (link) In brief, EOC cells (1.5 × 10⁵ cells per well in a 96-well plate) were seeded and washed in 200 μl of wash buffer (PBS/1% BSA) and incubated with 100 μl of wash buffer containing 1:500 of mouse anti-human monoclonal antibody against CAR (RmcB, Millipore, Watford, UK), 1:500 of CD46 (MEM-258, Abcam, Cambridge, UK) or mouse IgG control antibody (Santa Cruz Biotechnology, Heidelberg, Germany) for 1 hour on ice. Cells were washed three times and incubated with a 1:500 dilution of goat anti-mouse Alexa Fluor 647 antibody (Invitrogen, UK) for 1 hour on ice. Cells were fixed in 4 % paraformaldehyde (PFA) for a minimum of 10 min at 4 °C for flow cytometry. 2 × 10⁴ gated events were acquired in channel FL-4 on a BD Accuri C6 (BD Biosciences, USA) flow cytometer and data analysed in BD Accuri C6 software version 1.0.264.21 (Becton Dickinson, USA).

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