SMMC7721 and HepG2 cells were stained with anti-MICA/B (clone 6D4, Biolegend, CA, USA) for 30 min and fluorescein isothiocyanate (FITC)-labeled goat anti-mouse IgG1κ mAb (BD Biosciences, NJ, USA) for 20 min on ice. Mouse IgG1κ isotype control (BD Biosciences, NJ, USA) was used as a negative control. A minimum of 20,000 gated events/sample was collected on a flow cytometer (FACSCalibur, Elite ESP, FL, USA) and analyzed using CellQuest software. SMMC7721 and HepG2 cells from different treatment groups were lysed in lysis buffer (Bio-Rad, CA, USA), and the western blot analysis was performed as described previously [10 (link)]. The cytotoxicity analysis was performed with the CytoTox 96 Non-Radioactive LDH Cytotoxicity Assay (Promega, WI, USA), according to the protocol provided. To analyze the involvement of MICA/B in cytolytic activity of NK cells, anti-MICA/B mAb (6D4) or isotype-matched control Ab was added during the cytolytic assay.
Mouse igg1κ isotype control
Manufactured by BD
Sourced in United States
The Mouse IgG1κ isotype control is a laboratory reagent used as a control in flow cytometry, ELISA, and other immunoassays. It is a mouse monoclonal antibody of the IgG1 subclass with a kappa light chain, which serves as a negative control to establish background staining levels when detecting target analytes.
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Lab products found in correlation
Lysis buffer, by Bio-Rad (1 mentions)
Cytotox 96 non radioactive cytotoxicity assay ldh, by Promega (1 mentions)
Dynabeads sheep anti mouse igg, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by BD (1 mentions)
Lsrfortessa machine, by BD (1 mentions)
Ab11267, by Abcam (1 mentions)
Ab24170, by Abcam (1 mentions)
Anti c3, by Abcam (1 mentions)
Anti map2, by Abcam (1 mentions)
Anti lamp1, by Abcam (1 mentions)
Rat anti mouse cd11b, by BD (1 mentions)
Anti synaptophysin, by Abcam (1 mentions)
Anti gfap, by Cell Signaling Technology (1 mentions)
Anti iba1, by Abcam (1 mentions)
Anti psd95, by Abcam (1 mentions)
Alexa fluor 488 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Lsrfortessa x 20, by BD (1 mentions)
Cat 15710, by Electron Microscopy Sciences (1 mentions)
Pe conjugated anti cd43, by BD (1 mentions)
Recombinant human ifn γ, by Thermo Fisher Scientific (1 mentions)
9 protocols using mouse igg1κ isotype control
1
MICA/B Expression and NK Cell Cytotoxicity
2
CL-11 Monoclonal Antibody Sandwich ELISA
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Anti-CL-11 mAbs Hyb-15 and Hyb-17 (described in section Generation of Anti-CL-11 Monoclonal Antibodies and Development of a CL-11 Specific Sandwich ELISA), or mouse IgG1κ isotype control (BD Biosciences, USA) (2 μg each) were coupled to 25 μl of sheep anti-mouse IgG Dynabeads (Invitrogen) end-over-end for 30 min at RT. The conjugated beads were washed with PBS-Tw and incubated end-over-end 1 h at RT with serum diluted 1:1 in TBS-Tw-EDTA. After washing with PBS-Tw, bound CL-11 was eluted with 0.5% citric acid and subjected to SDS-PAGE as described below.
3
Flow Cytometry Analysis of Reprogramming Intermediates
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MEFs and reprogramming intermediates at days 3, 5, and 8 were dissociated with trypsin. Cells were collected by centrifugation at 300 × g for 5 min and kept on ice hereafter in polystyrene test tubes (12 × 75 mm, non-sterile). Cells were washed twice with 1 mL FACS buffer (1× PBS, 5% fetal bovine serum, 20 mM HEPES pH 7.2–7.5). Cells were resuspended and blocked in blocking buffer (FACS buffer containing 5% normal mouse serum (Gemini bio-products, #100–113)) at a density of 1 × 106 cells per 100 µL and pre-incubated on ice for at least 10 min. Cdh1 antibody (Thermo Fisher Scientific, #50−3249−82; 0.5 μg per sample, Supplementary Table 11 ) or Mouse IgG1, κ Isotype Control (BD biosciences, #555746; 5 μg per sample) was added directly to cells in blocking buffer and incubated for 20–25 min in dark on ice. The unbound antibodies were washed twice with 1 mL of cold FACS buffer. DAPI (BD biosciences, #564907) was used to exclude dead cells. Cells were passed through a 40 μm cell strainer (Corning, # CLS431750–50EA) before analysis on LSRFortessa machine (BD biosciences). The gating strategy is shown in Supplementary Fig. 2B . Analysis was done with the FlowJo V10 software.
4
Immunocytochemistry and Immunohistochemistry Antibodies
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The following primary antibodies were used in immunocytochemistry and immunohistochemistry studies: anti-C1q (Invitrogen, MA1-40311, lot TD2550204, 1:50); anti-C3 (Abcam, ab200999, lot GR294205-17, 1:200; Hycult, HM1065, lot 23152M1017-A, 1:50); anti-Iba-1 (Abcam, ab178847, lot GR3229566-2, 1:100; GeneTex, GTX101495, lot 41885, 1:50); anti-GFAP (Cell Signaling Technology, 3670S, lot 5, 1:100); anti-LAMP1 (Abcam, ab24170, lot GR3235359-1, 1:100); anti-MAP2 (Abcam, ab11267, lot GR281093-9, 1:100); anti-PSD95 (Abcam, ab12093, lot GR3271883-1; ab18258, lot GR3174013-1, 1:100); anti-Synaptophysin (Abcam, ab32127, lot GR312544-1, 1:100); 4G8 (Biolegend, 800704, lot B238676, 1:100); anti-β-actin (MBL, M177-3, lot 002, 1:1000); mouse anti-rat CD11b (BD Biosciences, 554980, lot 6294728); mouse IgA, κ isotype control (BD Biosciences, 553476, lot 7257918); mouse anti-rat CD32 (BD Biosciences, 550273, lot 8339705); mouse IgG1κ isotype control (BD Biosciences, 553447, lot 8241620).
5
Monocyte Modulation of T-Cell Response
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CD14+ monocytes were pretreated with 5 or 10 µg/mL anti-Dll-4 antibody (BD Biosciences) for 90 min prior to the addition of P. gingivalis LPS, and Mouse IgG1, κ Isotype Control (BD Biosciences) was used as the isotype control. The cells were then incubated with 1 µg/mL of P. gingivalis LPS for 24 h and were co-cultured with activated CD4+T cells in a 1:10 ratio for 5 days in a humidified atmosphere of 5% CO2 at 37 °C. Unblocked groups were used as controls.
6
Quantifying FcεRI Expression in Lung Cells
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The FcεRI expression was examined by flow cytometry. Five hundred thousand lung cells were incubated with the working dilution of purified mouse anti-rat high affinity IgE receptor (BD Biosciences, San Diego, CA, USA) or purified mouse IgG1, κ isotype control (BD Biosciences) in a
7
Exosome Immunophenotyping by Flow Cytometry
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MSC exosomes (3 × 109) were diluted in 200 μl of PBS, 10 μl of aldehyde latex beads (Invitrogen) added, and the samples rotated at room temperature for 15 min. Samples were diluted with 600 μl of PBS and incubated overnight at 4°C. 400 μl of 1 M glycine was added and the beads incubated for 30 min at room temperature, followed by centrifugation at 8,000g for 1 min. Beads were resuspended in 10% BSA in PBS, blocked for 1 h at room temperature, and centrifuged at 8,000g for 1 min. Samples were incubated with 2.5 μg/ml of primary antibody (CD9, SAB4700092; Sigma-Aldrich; CD47, 14-0479; eBioscience; CD63, 556019, BD; CD81, 555675, BD; mouse IgG1κ isotype control, 555746, BD) in 20 μl of 2% BSA in PBS for 1 h at room temperature. Samples were washed three times with 200 μl of 2% BSA in PBS, followed by incubation in 100 μg/ml Alexa Fluor 488 donkey anti-mouse IgG (A21202; Invitrogen) in 20 μl of 2% BSA for 1 h at room temperature. Samples were washed three times with 200 μl of 2% BSA in PBS and analyzed using a BD LSR Fortessa X-20. Data analysis was performed in FlowJo (BD) and positivity determined based on signal in the isotype control.
8
Bone Marrow Single-Cell Analysis
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Single-cell suspension from the BM of the experimental and control mice were prepared, followed by RBC lysis, and fixed with 2% PFA (Electron Microscopy Sciences Cat15710) at 37 °C for 10 min. Cells were washed twice with PBS and then slowly permeabilized with ice-cold 95% MeOH while vortexing on ice for 20 min. Cells were washed twice with PBS and incubated with Fc-block for 15 min on ice and then stained with PE/Cy7-conjugated anti-B220, PE-conjugated anti-CD43, AlexaFluor 647-conjugated anti-phospho-STAT5 (pY694, BD Biosciences), or mouse IgG1,κ isotype control (BD Biosciences). PE/Cy7-conjugated anti-B220 and PE-conjugated anti-CD43 were used at dilution of 1:400. AlexaFluor 647-conjugated anti-phospho-STAT5 was used at dilution of 1:100.
9
IFN-γ Regulates PD-L1 Expression
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Lk-2 and EBC-1 (obtained from the japanese Collection of Research Bioresource, Tokyo, japan) were maintained in a humidified atmosphere of 5% CO 2 at 37˚C. RPMI-1640 medium was used for Lk-2 culture, and EBC-1 cells were cultured in Dulbecco's modified Eagle's medium, each supplemented with 10% fetal bovine serum.
Flow cytometry. To examine the dose-dependent effect of IFN-γ on surface expression of PD-L1 on tumor cells, the cells were cultured in medium containing 0, 31.25, 125 or 500 pg/ml recombinant human IFN-γ (PeproTech, Rocky Hill, Nj, uSa) for 48 h. To examine the reversibility of IFN-γ-induced PD-L1 expression, the cells were cultured in the medium containing 1.0 ng/ml IFN-γ for 48 h.The culture medium was then replaced by fresh medium without IFN-γ, and cell culture was continued for an additional 48 h. Those cells were then harvested and stained with a phycoerythrin (PE)-labeled anti-human PD-L1 antibody (clone: MIH1) (BD Biosciences, San jose, Ca, uSa) or a mouse IgG1, κ isotype control (BD Biosciences). To examine IFN-γ receptor 1 expression on tumor cells, the cells were stained with a PE-labeled anti-human IFN-γ receptor 1 (CD119) antibody (clone: GIR-208) (eBioscience). Flow cytometric analysis was performed using BD FaCSCalibur, and the data were analyzed using BD CellQuest Pro software (BD Biosciences).
Flow cytometry. To examine the dose-dependent effect of IFN-γ on surface expression of PD-L1 on tumor cells, the cells were cultured in medium containing 0, 31.25, 125 or 500 pg/ml recombinant human IFN-γ (PeproTech, Rocky Hill, Nj, uSa) for 48 h. To examine the reversibility of IFN-γ-induced PD-L1 expression, the cells were cultured in the medium containing 1.0 ng/ml IFN-γ for 48 h.The culture medium was then replaced by fresh medium without IFN-γ, and cell culture was continued for an additional 48 h. Those cells were then harvested and stained with a phycoerythrin (PE)-labeled anti-human PD-L1 antibody (clone: MIH1) (BD Biosciences, San jose, Ca, uSa) or a mouse IgG1, κ isotype control (BD Biosciences). To examine IFN-γ receptor 1 expression on tumor cells, the cells were stained with a PE-labeled anti-human IFN-γ receptor 1 (CD119) antibody (clone: GIR-208) (eBioscience). Flow cytometric analysis was performed using BD FaCSCalibur, and the data were analyzed using BD CellQuest Pro software (BD Biosciences).
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