Ifenprodil

NMDA, NMDA receptor antagonists, ifenprodil and TTX were purchased from Tocris Bioscience (Bristol, UK). Media, media supplements, Lipofectamine 2000, fetal bovine serum, antibiotic/antimycotic and Β‐27 were purchased from Invitrogen (Carlsbad, CA, USA). The remaining substances were purchased from Sigma‐Aldrich (St Louis, MO, USA) unless otherwise stated.

The following commercial antibodies were used for Immunocytochemistry (ICC) and Western blot (WB) in the concentrations indicated: rabbit (rb) antibodies against GluN2B (alomone labs; ICC live staining: 1:200, fixed staining.: 1:1000; endocytosis assay: 1:25), antibodies against GluN2A (alomone labs; 1:500) and GluN1 (Synaptic Systems; 1:200), anti pGluN2BTyr1472 antibody (AAT Bioquest; WB 1:500); mouse (ms) antibodies against GluN2B (NeuroMab; WB 1:500), PSD-95 (NeuroMab; ICC 1:1000), Map2 (Sigma-Aldrich; ICC 1:2000), β3-tubulin (Synaptic System; WB 1:1000); rat antibody against β1-integrin CD29 (BD Pharmingen; 1:25).
Fluorescently labelled secondary antibodies that were used for ICC against rabbit, mouse, guinea pig were purchased from Invitrogen conjugated with either Alexa 488, 568, 647 (1:1000) or from Dianova conjugated with Cy3, Cy5 (1:1000). Fluorescently labelled secondary antibodies against ms, rb and guinea pig for quantitative immunoblotting were purchased from Invitrogen (ms Alexa Fluor 680, 1:20,000) and from Rockland (rb IRDye 800 W, 1:20,000).
Hyaluronidase (Hya, Sigma-Aldrich) was used at 100 units/ml, and TTX (0, 5 µM), CNQX (5 µM), Biccuculine (BCC) (10 µM), AP5 (10 µM), Ifenprodil (3 µM) was purchased from Tocris. All other chemicals and drugs were purchased from Sigma-Aldrich (USA).

D-(-)-2-AMINO-5-PHOSPHONOPENTANOICACID (D-AP5) and Ifenprodil (NR2B specific antagonist) were obtained from Tocris Bioscience,(Avonmouth, Bristol, UK). Isoproterenol, Gabazine, and 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) were purchased from Sigma Aldrich(St.Louis, MO,USA).

Apoptosis was determined with the Apoptosis detection kit from BD
Pharmingen (Heidelberg, Germany). 2×10⁵ splenic B cells
were left untreated or were activated with α-IgM (10 μg/ml) or LPS (10 μg/ml)
without or with costimulation by CD40 Abs (5 μg/ml, Biolegend, San Diego, CA, USA)
in the presence or absence of ifenprodil (30 μM, Tocris Biosciences). At 24 h of
culture cells were harvested, stained with Annexin V-FITC (BD Pharmingen) and
propidium iodide (PI, Sigma-Aldrich) according to manufacturer’s protocol and
analyzed by flow cytometry using a FACSFortessa and Cell Quest software (BD
Biosciences). The percentage of viable cells was determined by gating on
AnnexinV⁻PI⁻ cells.

Splenic B cells were isolated with the B-cell isolation kit from
Miltenyi Biotech (Bergisch Gladbach, Germany) according to the manufacturer’s
protocol. Purity of B cells was 90-95%. B cells were activated with α-IgM
(10 μg/ml, Jackson Immunoresearch Laboratories, Hamburg, Germany),
lipopolysaccharide (LPS, 10 μg/ml, E. coli 0111:B4, Sigma-Aldrich, Taufkirchen,
Germany), or PMA (100 ng/ml, Calbiochem, Darmstadt, Germany) and IO (700 ng/ml,
Sigma) in complete RPMI1640 medium (Biochrom AG, Berlin, Germany) supplemented
with 10% FCS, 50 μM β-mercaptoethanol, 1% penicillin/streptomycin. NMDAR
antagonist ifenprodil, memantine, or D-APV (diluted in
ddH₂O, all from Tocris Biosciences, Bristol, Great Britain)
were added in concentrations as given. Proliferation was measured at 24 h of
culture by ³[H]-Thymidine incorporation (0.2 μ Ci/well,
MP Biochemicals Europe, Heidelberg, Germany) for 16 h.

CNQX, MK-801, ifenprodil, and Ro25-6981 were obtained from Tocris Cookson (Bristol, UK), and picrotoxin was obtained from Sigma-Aldrich (St. Louis, MO). NVP-AAM077 (NVP) was obtained from Professor Yao’s lab at the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences.