The hybridoma was prepared following the manufacturer’s instructions (Roche, Basel, Switzerland) with minor modifications. Briefly, the mouse spleen cells were mixed at a ratio to Sp2/0-Ag14 of 5:1 (ATCC, VA, USA) in a sterile 50-ml conical tube, which was centrifuged to pellet the cells at 800 rpm for 10 minutes. After discarding the supernatant, 1 ml of 50% PEG 1500 (Roche) was slowly added to the cell pellet dropwise over a 1-minute period and the cells were swirled for 90 seconds in a 37 °C water bath. Cell fusion was stopped by adding Roswell Park Memorial Institute medium (RPMI) 1640 (Gibco, CA, USA) containing 10% fetal bovine serum (FBS, Invitrogen, CA, USA) with gentle swirling at RT for 10 minutes. After washing with RPMI 1640 twice, cells were suspended in 30 ml of RPMI1640 supplemented with 10% FBS, 10% BM Condimed H1 (Roche) and 1x HAT (Gibco), plated 2.5 ml per well in a 6-well culture dish and incubated at 37 °C in a 5% CO2 incubator. Limiting dilution was carried out for selection of a single colony, which was amplified in RPMI 1640 supplemented with 10% FBS, 10% BM Condimed H1 (Roche), 1× HT (Gibco) and 1x hybridoma fusion & cloning supplement (Roche). The supernatant was harvested for ELISA of antibody activity to pp65422-439.
Bm condimed h1
Manufactured by Roche
Sourced in United States
The BM Condimed H1 is a laboratory equipment product manufactured by Roche. It is designed for use in clinical and research laboratory settings. The core function of the BM Condimed H1 is to perform condensation measurements, which are essential in various analytical and diagnostic applications. The product specifications and technical details are not available in this response.
Automatically generated - may contain errors
Lab products found in correlation
Peg1500, by Roche (2 mentions)
Sp2 0 ag14, by American Type Culture Collection (1 mentions)
Rpmi 1640, by Roche (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
P3u1 cells, by American Type Culture Collection (1 mentions)
Mdckii, by Nichirei Biosciences (1 mentions)
Penicillin, by Nacalai Tesque (1 mentions)
Streptomycin, by Nacalai Tesque (1 mentions)
Streptomycin, by Roche (1 mentions)
Penicillin, by Roche (1 mentions)
Protein assay bca kit, by Nacalai Tesque (1 mentions)
Hybridoma sfm medium, by Thermo Fisher Scientific (1 mentions)
Amicon ultra 100k, by Merck Group (1 mentions)
Hybridoma sfm, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Fujifilm (1 mentions)
Streptomycin, by Fujifilm (1 mentions)
Fetal bovine serum (fbs), by MP Biomedicals (1 mentions)
Polyethylene glycol 2000, by Merck Group (1 mentions)
4 pba, by Merck Group (1 mentions)
7 protocols using bm condimed h1
1
Hybridoma Production and Antibody Screening
2
Stable cell lines expressing claudins
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HT-1080 cells stably expressing mock (empty vector), human CLDN-1 to -7 and mouse CLDN-5 were developed as described previously32 . MDCKII and P3U1 cells were purchased from ATCC (Manassas, VA). HT-1080 cells expressing human CLDN-5 mutants (D68E, T75A and S151T) and MDCKII cells expressing human or mouse CLDN-5 were prepared in a similar way as described previously32 .
HT-1080 and MDCKII cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (v/v) (Nichirei Biosciences, Tokyo, Japan), 100 U/mL penicillin, and 100 µg/mL streptomycin (Nacalai Tesque, Kyoto, Japan). P3U1 cells were maintained in RPMI1640 medium supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. All hybridomas were maintained in 20% heat-inactivated FBS, 10% BM Condimed H1 (Roche Diagnostics), 100 U/mL penicillin, and 100 µg/mL streptomycin. All cells were incubated under an atmosphere of 5% CO2 in air at 37 °C.
HT-1080 and MDCKII cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (v/v) (Nichirei Biosciences, Tokyo, Japan), 100 U/mL penicillin, and 100 µg/mL streptomycin (Nacalai Tesque, Kyoto, Japan). P3U1 cells were maintained in RPMI1640 medium supplemented with 10% heat-inactivated FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin. All hybridomas were maintained in 20% heat-inactivated FBS, 10% BM Condimed H1 (Roche Diagnostics), 100 U/mL penicillin, and 100 µg/mL streptomycin. All cells were incubated under an atmosphere of 5% CO2 in air at 37 °C.
3
Purification of Anti-CLDN Monoclonal Antibodies
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Purified rat anti-CLDN-5 mAbs (clone R9) and mouse anti-CLDN-1 mAbs (clone 2C1) were prepared as described previously [49 (link),50 (link)]. Briefly, hybridoma cells were cultured in Hybridoma SFM medium (Thermo Fisher Scientific, Waltham, MA, USA) containing 10% BM Condimed H1 (Roche, Mannheim, Germany). The anti-CLDN mAbs were purified from the culture media using Protein G Sepharose 4 Fast Flow columns (Cytiva, Marlborough, MA, USA). Buffer exchange was performed using phosphate-buffered saline (PBS, pH 7.4) and centrifugal filter tubes with a molecular weight cutoff of 100 kDa (Amicon Ultra-100K, Merck Millipore, Burlington, MA, USA). Purified mAb was sterilized by filtering it through a 0.22 µm filter, and then stored at −30 °C. The concentration of mAb was quantified using a BCA Protein Assay kit (Nacalai Tesque, Kyoto, Japan) with bovine serum albumin as the standard.
4
Generation of Anti-SDF-2 Rat Monoclonal Antibodies
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Anti-SDF-2 rat mAbs were generated using the rat lymph node method established by Sado et al.(12 (link)) A 9-week-old female WKY/Izm rat (Japan SLC, Hamamatsu, Japan) was injected in the hind footpads with 200 μL of an emulsion containing 266 μg of a synthetic peptide of 20 amino acids (GIFMKPSELLKAEAHHAELC), which corresponded to residues 193–211 of human SDF-2, and complete Freund's adjuvant. After 19 days, the cells from the medial iliac lymph nodes from the immunized rat were harvested and then fused with mouse myeloma SP2 cells at a ratio of 5:1 in a 50% polyethylene glycol (PEG 1500; Roche, Basel, Switzerland) solution. The resulting hybridoma cells were plated into 96-well plates and cultured in HAT selection medium (Hybridoma-SFM [Thermo Fisher Scientific]; 10% FBS; 5% BM-condimed H1 [Roche]; 100 mM hypoxanthine; 0.4 mM aminopterin; and16 mM thymidine). Eight days postfusion, the hybridoma supernatants were screened using an enzyme-linked immunosorbent assay (ELISA) against the human SDF-2 synthetic peptide. Positive clones were subcloned and rescreened by ELISA, immunoblotting, and immunoprecipitation.
5
Hybridoma Cell Line Isolation and Culture
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A hybridoma cell line (B5F6, anti-mouse IL-17RB mAb-producing line) has been established as described previously (27 (link)) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS (MP Biomedicals), 10% BM Condimed H1 (Roche), and penicillin (100 U/ml) and streptomycin (100 μg/ml; Wako Chemicals) in a cell incubator at 37°C and 5% CO2. The cells were subcultured every 3 days at 8 × 104 cells/ml in 10 ml of medium in dishes or flasks and used for sorting experiments 3 days after the last passage in the mid-logarithmic phase. To sort the hybridoma cells, the cells were stained by the same procedure as the staining of Jurkat cells and resuspended in the cell culture medium containing 10% FBS at a final concentration of 7 × 106 cells/ml.
6
Monoclonal Antibody Generation Against HTLV-1 HBZ
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To produce anti-HBZ mAbs, C57BL/6 mice or WKAH rats were immunized three or four times at 2-week intervals with keyhole limpet hemocyanin (KLH)-conjugated peptide fragments of HBZ (i.e., Peptides #1, #2, and #3; Additional file 1 : Figure S1A) or histidine-tagged recombinant HBZ, which was produced in a wheat germ extract-based cell-free transcription and translation system (WEPRO7240H Expression kit; CellFree Sciences, Japan) (Additional file 1 : Figure S1B). It is important to note that hbz gene is the only HTLV-1 gene with no nonsense mutation and it is genetically highly conserved among isolates, since human APOBEC3G generates nonsense mutations in the plus-strand coding sequence of HTLV-1 proviral genomes in vivo, targeting the minus strand of HTLV-1 during reverse transcription [6 (link)]. After the booster immunization, mouse or rat spleen cells were isolated and fused with mouse myeloma cells (Sp2/0-Ag14) using 50 % polyethylene glycol 2000 (Merck, Darmstadt, Germany). After selection in hypoxanthine–aminopterin–thymidine (HAT) medium, cells were cloned by limiting dilution (1 cell per well) with BM Condimed H1 (Roche, Indianapolis, IN), at least twice to ensure monoclonality. Individual clones growing in particular wells were tested by ELISA with the immunizing antigens.
7
Generation of Anti-NL4X Antibodies
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The following antibodies were used: HA-high (3F10, Roche, ×2000 dilution), α-tubulin (DM1A, SIGMA, ×2000 dilution), βIII-tubulin (TUJ1, SIGMA, ×5000 dilution), VGAT (#131002, Synaptic Systems, ×1000 dilution), vGlut1 (#135303, synaptic systems, ×1000 dilution), V5 tag (R960-CUS, Invitrogen, ×5000 dilution) ADAM10 (ab1997, abcam, ×500 dilution). For rabbit polyclonal antibody, SAJ520206 was raised against synthetic peptide corresponding to NL4X cytoplasmic region (723-741) by SIGMA. For rat monoclonal antibody against extracellular region of NL4X, we injected 250 μg of the recombinant human NL4X protein (5158-NL, R&D Systems) with Freund’s adjuvant complete (SIGMA) into the foot pad of WKY/Izm rat. After three additional immunization with Freund’s adjuvant incomplete (SIGMA), iliac and inguinal lymph nodes were obtained. B cells were fused with PAI cells (JCRB0113) by polyethylene glycol (Roche) and cultured with GIT medium containing 5% FBS, hypoxanthine/aminopterin/thymidine (SIGMA), and BM Condimed H1 (Roche). Screening was performed by immunocytochemical analysis using HEK293 cells stably expressing NLs. After limiting dilution and further screening, we selected clone 2C3 as human NL4X specific rat monoclonal antibody. 4PBA was purchased from SIGMA. INCB3619 was synthesized according to the patent descriptions as previously described [17 ].
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