The presence of HLA class I and II on the surface of spermatozoa was assessed by direct immunofluorescence using a BD FACSCalibur (BD Biosciences, USA) flow cytometer. Two tubes for each sample containing 1 × 106 spermatozoa were prepared. One was incubated with phycoerythrin (PE) mouse anti‐human HLA‐ABC (clone: G46‐2.6, BD pharmingen, USA) and the other was incubated with phycoerythrin (PE) mouse anti‐human HLA‐DR (clone: G46‐6, BD pharmingen, USA) at room temperature for 30 min. After two washes with AllGrad Wash (400 g for 5 min), tubes were run through the flow cytometer. Data from at least 100,000 events were collected using forward scatter (a logarithmic amplifier) and side angle of light scatter (a logarithmic amplifier). As a negative control, we used unstained control. Isotype controls [Mouse IgG1, κ (clone: G46‐2.6, BD pharmingen, USA) and Mouse IgG2a, κ (clone: G46‐6, BD pharmingen, USA)] were used to determine background fluorescence (autofluorescence and non‐specific binding of antibodies). To remove background fluorescence, antibody titration was done and the optimal titer that showed minimum background was selected. Noteworthy, cell viability test was not performed because we removed abnormal and dead spermatozoa by AllGrad solution before staining. Fluorescence data were obtained with the logarithmic amplifier. We used flowJo vx software for data analyses.
Mouse igg1κ
Manufactured by BD
Sourced in United States
Mouse IgG1κ is an antibody isotype commonly used in research applications. It serves as a control or reference for immunoassays and other immunological techniques.
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Lab products found in correlation
Pe mouse anti human hla abc, by BD (1 mentions)
Pe mouse anti human hla dr, by BD (1 mentions)
Facscalibur, by BD (1 mentions)
Canto flow cytometer, by BD (1 mentions)
T2200, by Merck Group (1 mentions)
Streptavidin pe, by Thermo Fisher Scientific (1 mentions)
Mouse igg2b, by BioLegend (1 mentions)
Attune classic flow cytometer, by Thermo Fisher Scientific (1 mentions)
Epcam, by Sony (1 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Cd271, by BD (1 mentions)
Mouse igg2b, by R&D Systems (1 mentions)
Ec800 flow cytometer, by Sony (1 mentions)
Rat igg2a, by R&D Systems (1 mentions)
Anti tim 3 apc, by R&D Systems (1 mentions)
Anti nkg2d apc, by BD (1 mentions)
Facsverse, by BD (1 mentions)
Nupage novex system, by Thermo Fisher Scientific (1 mentions)
Mouse anti human cd14 fitc, by BD (1 mentions)
Mouse igg2b κ, by BD (1 mentions)
11 protocols using mouse igg1κ
1
HLA Expression on Human Spermatozoa
2
Immunophenotyping of Neural Progenitors
3
Characterizing Melanoma Heterogeneity
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Primary tumors, lymph nodes, and lungs were enzymatically digested into single-cell suspensions to characterize melanoma heterogeneity as described above. Erythrocytes were lysed with 1 × ACK buffer (1.5 M NH4Cl, 100 mM NaHCO3, 10 mM EDTA) and washed with PBS. Cells from primary tumors, lymph nodes, lungs, and blood were stained for viability using the LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (L34957, Invitrogen™, Carlsbad, CA) for 20 min at 4 °C. After washing with PBS, the cells were stained with a cocktail of primary antibodies or corresponding isotype controls, including EpCAM (2221100, SONY, Tokyo, Japan), Trop2 (1B-898-C100, Exbio, Vestec, Czech Republic), and CD271 (562122, BD Biosciences, San Jose, CA) (Supplementary Tables 1 and 2 ), as well as mouse IgG2b (400342, Biolegend, San Diego, CA), and mouse IgG1 κ (557872, BD Biosciences, San Jose, CA). After 20 min of incubation at 4 °C, staining with Streptavidin PE (12-4317, eBioscience, San Diego, CA) was performed for 20 min at 4 °C. The gating of positive populations was done using isotype controls. Only viable single GFP+ cells without debris were included in the analysis. The samples were measured on an Attune Classic flow cytometer (ThermoFisher Scientific, Waltham, MA) and analyzed using FlowJo software.
4
Evaluating Cell Surface Antigen Expression
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Cell surface antigen expressions were analyzed using flowcytometry. The cells were incubated with anti-HER2-PE (clone; #191924, isotype; mouse IgG2b, R&D Systems, MN), anti-NKG2D-APC (clone; 1D11, isotype; mouse IgG1κ, BD Biosciences, Heidelberg, Germany), anti-Tim3-APC (clone; #344823, isotype; rat IgG2a, R&D systems) for 30 min at 4 °C. The cells were then washed and analyzed using an EC800 flowcytometer (SONY, Tokyo, Japan).
5
Immunophenotyping of Human Endothelial Cells
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HUVECs were grown in 12-well plates as described above. Thereafter, supernatant was aspired; the cells were washed with warm sterile PBS and de-attached with TrypLE. The reaction was stopped with complete EGM medium, the cells aspired and transferred into FACS tubes and centrifuged at 1200 rpm for 7 minutes at RT. Then, the cells were then incubated with Fc-blocking solution (anti-CD16/anti-CD32) (eBioscience, Cat. No. 14-0161-81C) for 10 minutes at +4°C. Thereafter, rabbit-anti-human HSP60 (Santa Cruz Biotechnologies, Cat. No. sc-13966, clone H-300) at 1:25 dilution together with a monoclonal mouse-anti-human CD31 FITC antibody (BD, Cat. No. 555445) at 1:20 dilution in 2%FCS/PBS was added and cells incubated for 30 minutes at +4°C. After washing with 2%FCS/PBS, the secondary donkey-anti-rabbit A568 (Abcam, Cat. No. ab175470) was added at 1:200 dilution, and incubated for 30 minutes at +4°C. After a final washing step, the cells were re-suspended in 2%FCS/PBS and acquired on a FACS Verse (BD Biosciences). The results were analyzed using FlowJo. As a negative control, secondary alone or mouse IgG1κ (BD, Cat. No. 51-35405X) were used.
6
Investigating Anti-C2 mAb Mechanism
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Example 9
To investigate the mechanism of inhibition of the anti-C2 mAbs, glycosylated recombinant human C2 was pre-incubated with mAbs anti-C2-5F2.4, anti-C2-13, anti-C2-32, anti-C2-35 and anti-C2-60 (at a molar C2-to-antibody ratio of 1:2) for 30 min at RT. In parallel, isotype controls of mouse IgG1κ and IgG2aκ (both from BD Biosciences) were tested as negative controls. Then, human C1s (C1s-to-C2 ratio of 1:25; Calbiochem) was added for 1 hour at 37° C. The mixtures were analyzed by SDS-PAGE using the pre-cast gel NuPage® Novex® system (Invitrogen), and stained with Coomassie brilliant blue.
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7
Psoriasin Inhibits Bacterial Growth
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Human uroepithelial cells, TERT-NHUC were treated with 6 or 30 mM glucose. After 24 h of treatment, medium was removed, and cells were lysed in 1% Triton X‐100 in PBS. Cell free supernatant was obtained by centrifugation at 8000 g for 5 mins and then incubated for 30 mins at 37 °C with 1 µg/ml of monoclonal mouse anti‐psoriasin antibody (Santacruz Biotechnology) or the same concentration of an isotype control antibody (mouse (Ig)G1,κ; BD Biosciences). Bacteria were prepared as above and 50 µl from 104 CFU/ml bacterial suspension was added to 150 µl of pretreated cell lysate. After incubation for 30 min at 37 °C, 100 µl aliquots were plated and bacterial survival was determined by viable count. Results were expressed in relation to control cell lysates pretreated with control antibodies.
Urine samples, with no bacterial growth from nondiabetic controls and T2D patients were collected, and E. coli CFT073 was prepared as above and 50 µl from 104 CFU/ml bacterial suspension was added to 150 µl of urine with 5 µM of psoriasin peptide. After incubation for 30 min at 37 °C, 100 µl aliquots were plated and bacterial survival was determined by viable count.
Urine samples, with no bacterial growth from nondiabetic controls and T2D patients were collected, and E. coli CFT073 was prepared as above and 50 µl from 104 CFU/ml bacterial suspension was added to 150 µl of urine with 5 µM of psoriasin peptide. After incubation for 30 min at 37 °C, 100 µl aliquots were plated and bacterial survival was determined by viable count.
8
Monocyte-Macrophage Differentiation Assay
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The activity of THP-1 monocytic cells (their differentiation into macrophages without PMA but only in the presence of the studied plates) was checked by expression of CD 14 and CD 68 antibodies. The expression of CD14 and CD68 were evaluated by flow cytometry using mouse anti-human CD14 FITC and mouse anti-human CD64 Alexa Fluor 647 clone Y1:82A antibody (BD Pharmingen, San Diego, CA, USA). Cells were compared to an isotype control, mouse IgG1 κ and mouse IgG2b κ (BD Pharmingen). Briefly, the cells were stained in phosphate-buffered saline (PBS, Ca2+- and Mg2+-free) supplemented with 2% bovine calf serum (BCS, Hyclone, Logan, UT, USA). After the final wash, cells were re-suspended in PBS and analyzed by FACS using the Navios flow cytometer (Beckman Coulter, Brea, CA, USA).
9
Characterization of MSC Phenotype
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The third-passage cells were harvested and washed 3 times with PBS. Then, a single-cell suspension was obtained by digestion with 0.25% trypsin. The cells were resuspended with PBS-1% fetal bovine serum (FBS) after centrifugation. Next, 1×106 cells were incubated in the dark for 30 min at 4°C with the corresponding commercial monoclonal antibodies (CD29, cloneTS2/16, BioLegend, Ref.303003, 1: 20; CD90, clone5E10, BD Pharmingen™, Ref.555596, 1: 20; CD166, clone3A6, Ancell Corporation, Ref.393–040, 1: 50; CD45, cloneHI30, BD Pharmingen™, Ref.555483, 1: 5; CD34, clone581, BD Pharmingen™, Ref.555822, 1: 5; Isotype controls: Mouse IgG1, AbD Serotec, Ref.MCA928F, 1: 10; Mouse IgG1, κ, BD Pharmingen™, Ref.555749, 1: 5). Flow cytometry was performed on a FACSCanto device (BD Biosciences). The data were analyzed using FACSDiva (BD Biosciences) and FlowJo software (TreeStar, Inc, Ashland, OR).
10
OVA-H-2Kb Presentation and T Cell Activation
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To investigate OVA-H-2Kb production, BM-DCs were incubated for 24 h with PBS, OVA, or rlipo-OVA and stained with CD16/CD32 (clone 2.4G2, BD Biosciences) prior to cell surface staining with the CD11c-FITC (clone N418, Biolegend, San Diego, CA, USA) and SII/H-2Kb-PE (clone 25-D1.16, eBioscience) antibodies and the isotype control antibody (mouse IgG1κ, BD Biosciences). Then, the cells were subjected to intracellular SII/H-2Kb or isotype control antibody staining using the Intracellular Fixation & Permeabilization Buffer Set according to the manufacturer's instructions (eBioscience). To confirm the presentation of SII/H-2Kb could activate T cells, BM-DCs (1 × 104) were cultured with 1 × 105 OT-I cells (purity > 90%) which were purified by CD8a+ T cell isolation kit (Miltenyi Biotec, Auburn, CA, USA), 1 μCi/well [3H]thymidine (Perkin-Elmer Life Science, Boston, MA) was added at 54 h, [3H]thymidine incorporation was measured at 72 h of culture, and performing scintillation counting. The level of IFN-γ was determined using ELISA kits (eBioscience) on day 5.
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