The following materials are used in the cell culture: L-leu (#L8912), lysine (Lys, #L9037), arginine (Arg, #A6969), Collagen Ⅰ (#SCR103), Basic Fibroblast Growth Factor (bFGF, #13256-029), BSA (A1933), and DMSO (#C6295), which were purchased from Sigma-Aldrich, USA. DMEM (#11995065), L-leu-deprived DMEM powder (#88425), fetal bovine serum (FBS, #10099141C), Antibiotics-antimycotic (#15240062), and Dulbecco phosphate-buffered saline (DPBS, #14190144) were purchased from Gibco, Life, Technologies, Grand Island, NY, USA. Culture dish (10 and 6 cm), Cell ware 6, 24, and 96-well plates, Matrigel and cell strainer, were purchased from Corning, NY, USA. Accutase, Cell Detachment Solution was purchased from innovative cell technologies, SD, USA. Mouse monoclonal anti-PAX7 (#sc-81648) was purchased from Santa Cruz Biotechnology, CA, USA. Donkey anti-Mouse IgG (H + L) was purchased from Invitrogen (#A10036, Invitrogen, Carlsbad, CA, USA). DAPI (#C0065), Triton X-100 (#9002-93-1), and Tween 20 (#9008-64-5) were purchased from Solarbio (Beijing, China). CCK 8 was purchased from Good Laboratory Practice Bioscience, Montclair, CA, USA.
Dulbecco phosphate buffered saline dpbs
Manufactured by Thermo Fisher Scientific
Sourced in United States
Dulbecco's phosphate-buffered saline (DPBS) is a balanced salt solution commonly used in various laboratory applications. It is a sterile, isotonic buffer that helps maintain the pH and osmotic environment for cells and tissues in vitro. DPBS is available in different formulations and is used to wash, suspend, and dilute samples during experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Donkey anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
Antibiotics antimycotic, by Thermo Fisher Scientific (1 mentions)
L leu, by Merck Group (1 mentions)
Collagen 1, by Merck Group (1 mentions)
Mouse monoclonal anti pax7, by Santa Cruz Biotechnology (1 mentions)
Accutase cell detachment solution, by Innovative Cell Technologies (1 mentions)
Basic fibroblast growth factor bfgf, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Tween 20, by Solarbio (1 mentions)
Cell strainer, by Corning (1 mentions)
Triton x 100, by Solarbio (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Anti cd28 clone 37, by BD (1 mentions)
Retronectin, by Takara Bio (1 mentions)
Anti cd3ε clone 145 2c11, by BD (1 mentions)
5 protocols using dulbecco phosphate buffered saline dpbs
1
Optimized Cell Culture Protocol for PAX7 Analysis
2
Fabrication of BNC/GelMA Hydrogels
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BNC was produced using BC as previously described[30 ]. In brief, BC was purified with 2% (w/v) NaOH at 80°C for 1 h and then washed repeatedly with distilled water until a neutral pH was obtained, was repeatedly hydrolyzed and centrifuged under acidic conditions to obtain BNC suspension, which was then dialyzed and freeze-dried to produce BNC powder. GelMA lyophilized powder and LAP were dissolved in Dulbecco phosphate-buffered saline (DPBS; Gibco, Grand Island, NY, USA) to form 10% w/v GelMA and 0.25% w/v LAP-based solutions, which were used in the control group. A certain proportion of irradiated BNC was added to the GelMA solution to form BNC/GelMA hydrogels with different concentrations (Table 1 ).
3
HLA Antibody Screening by Luminex SAB
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Initial testing of 10 lots, neat and adsorbed to remove common interfering agents and/or antibodies which might react with the polystyrene matrix of the SAB (Adsorb Out, One Lambda Thermo Fisher, Canoga Park, CA), showed a drop in the negative control MFI values.
Prior to further testing, all lots were treated with Adsorb Out™ according to manufacturer's instructions. Adsorbed H-Ig was then diluted with Dulbecco phosphate-buffered saline (D-PBS) (Gibco Life Technologies, Grand Island, NY) to 1:10 as our baseline working solution and in some experiments was further diluted 1:2, 1:5, and 1:10.
HLA IgG antibodies were determined by Luminex SAB (One Lambda Thermo Fisher and Immucor Lifecodes, Stamford, CT), according to the manufacturer's instructions. Immunofluorescence was determined using a Luminex LABScan 100 machine (Luminex Corp, Austin, TX) and results exported for analysis with the appropriate manufacturer's software (HLAFusion, One Lambda Thermo Fisher; MatchIT, Immucor Lifecodes).
Prior to further testing, all lots were treated with Adsorb Out™ according to manufacturer's instructions. Adsorbed H-Ig was then diluted with Dulbecco phosphate-buffered saline (D-PBS) (Gibco Life Technologies, Grand Island, NY) to 1:10 as our baseline working solution and in some experiments was further diluted 1:2, 1:5, and 1:10.
HLA IgG antibodies were determined by Luminex SAB (One Lambda Thermo Fisher and Immucor Lifecodes, Stamford, CT), according to the manufacturer's instructions. Immunofluorescence was determined using a Luminex LABScan 100 machine (Luminex Corp, Austin, TX) and results exported for analysis with the appropriate manufacturer's software (HLAFusion, One Lambda Thermo Fisher; MatchIT, Immucor Lifecodes).
4
Immunological Reagent Characterization for T-cell Studies
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For cell stimulation, anti‐CD3ε (clone 145‐2C11) and anti‐CD28 (clone 37.51) antibodies were purchased from BD Biosciences. Antibodies against p‐(Y701) STAT1 (clone 58D6), p‐(Y694) STAT5 (D47E7) XP® and STAT1 were from Cell Signaling. Anti‐actin (clone ACTN05) was purchased from Thermo Fisher Scientific. The mouse antibody against PTPN2 (clone 6F3) was provided by M. Tremblay (McGill University). For immunofluorescence staining and immunohistochemistry, rabbit anti‐CD3ε and mouse anti‐HER‐2 from Abcam were used. Recombinant human IL‐2, murine IL‐7 and IL‐15 used for T‐cell stimulation or IFNγ used for stimulating tumour cells were purchased from the NIH or PeproTech, respectively. RetroNectin was purchased from Takara, Dnase I and doxycycline from Sigma‐Aldrich. The mouse anti‐nuclear antibodies Ig's (total IgA+G+M) ELISA Kit (Alpha Diagnostic Int.) and the Transaminase II Kit (Wako Pure Chemicals) were used according to the manufacturer's instructions. FBS was purchased from Thermo Scientific; Dulbecco‐Phosphate‐Buffered Saline (D‐PBS), RPMI 1640, DMEM, MEM non‐essential amino acids and sodium‐pyruvate were from Invitrogen, and collagenase type IV was purchased from Worthington Biochemical.
5
Spheroid Culture and Inhibitor Treatment Protocol
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Briefly, Nanoculture Media-50M (NCM-50M; B-Bridge International, Cupertino, CA) containing 10% FBS and 1% penicillin, streptomycin, and amphotericin was heated to 37°C and 500 μl was plated into each well of a 24-well, low-affinity binding, square or honeycomb Nanoculture plate (B-Bridge International). The plates were spun at 500g for 3 minutes. Afterward, cells were trypsinized and suspended in 500 μl of NCM-50M and added to each well. Cells were allowed to form spheroids for 2 to 4 days. Subsequent to spheroid formation, cells were treated with pharmacological inhibitors or vehicle for 8 to 18 hours. At the end of treatment, spheroids were harvested from each well with gentle pipetting, spun, and washed with 1 × Dulbecco Phosphate-Buffered Saline (DPBS, Invitrogen) containing 10 mM sodium fluoride and 1 mM sodium orthovanadate. Cell pellets were subsequently lysed with modified NP-40 lysis buffer described above and protein was collected.
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