A total of 4×105 PBMCs were stimulated in a 96-well flat-bottom tissue culture plate (Costar, Corning, NY, USA) at 37°C in the presence of media, 15µg/mL anti-canine CD3 (AbD Serotec, Raleigh, NC, USA), or 0.25µg/mL concanavalin A (Sigma-Aldrich) for the indicated days. For assessment of proliferation, PBMCs were washed twice with PBS, incubated with 5µM CFSE (Molecular Probes, Eugene, OR), quenched with FCS, and plated. Two days post-stimulation, centrifuged and washed cells were incubated with or without CCL19-hIg (ELC; (Hargreaves et al., 2001 (link))) supernatant at 4°C for 45 min. Antibodies used were anti-CD8-Pacific Blue, anti-CD62L-PE (AbD Serotec), anti-human IgG-AF488 (Molecular Probes), and a cocktail containing anti-dog Pan T cell-APC, anti-B cell-PE, and anti-dog T cell Activation marker-FITC (Dog Activated T Lymphocyte Cocktail, BD Pharmingen, BD Biosciences, San Jose, CA). 7-amino-actinomycin D (7AAD; BD Pharmingen) was included for live/dead cell discrimination. Cells were stained in PAB for 45 min at 4°C, washed, and fixed in 2% formaldehyde.
Anti human igg af488
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Anti-human IgG-AF488 is a fluorescently-labeled secondary antibody that binds to human immunoglobulin G (IgG) antibodies. It is designed for use in various immunoassay and cell-based applications requiring the detection of human IgG.
Automatically generated - may contain errors
Lab products found in correlation
Anti cd62l pe, by Bio-Rad (2 mentions)
Hoechst 33342 dna stain, by Thermo Fisher Scientific (2 mentions)
Metaxpress software, by Molecular Devices (2 mentions)
Imagexpress pico automated cell imaging system, by Molecular Devices (2 mentions)
7 aminoactinomycin d, by BD (1 mentions)
96 well flat bottom tissue culture plate, by Corning (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Concanavalin a (cona), by Merck Group (1 mentions)
Anti canine cd3, by Bio-Rad (1 mentions)
Sodium azide, by Merck Group (1 mentions)
Chamber slide, by Ibidi (1 mentions)
Confocal microscope, by Olympus (1 mentions)
Anti rab5, by Cell Signaling Technology (1 mentions)
Anti rabbit igg af488, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg af568, by Thermo Fisher Scientific (1 mentions)
Anti tomm20 11802 1 ap, by Proteintech (1 mentions)
6 protocols using anti human igg af488
1
Canine PBMC Proliferation and Activation Assay
2
Flow Cytometric Analysis of PBMC Subsets
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PBMCs (500 000) were incubated in complete RPMI 1640 supplemented with 10% heat-inactivated FCS for 1 h at 37 °C, centrifuged, and resuspended in 100 μL of CCL19-hIg (ELC; [33 (link)]) culture supernatant for 45 min at 4 °C. After incubation, cells were washed and resuspended for staining in PAB solution containing PBS with 1% BSA and 0.05% sodium azide (both from Sigma-Aldrich). Antibodies used were anti-CD8 PacBlue, anti-CD4 AF647, anti-CD62L PE (AbD Serotec), CTL2.58-PeCy7, and anti-human IgG AF488 (Molecular Probes, Eugene, OR, USA). 7-amino-actinomycin D (7AAD, BD Pharmingen, BD Biosciences, San Jose, CA, USA) was included for live/dead cell discrimination. Following incubation of PBMCs with antibody mixes for 45 min on ice, cells were washed twice, fixed in 2% formaldehyde, and analyzed by flow cytometry.
3
Antibody-Mediated Dengue Virus Internalization
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Antibodies (1 μg/ml) were incubated with DENV-1 (or DENV-2 [data not shown]) for 1 h at 37°C. BHK-21 cells were grown on chamber slides (ibidi). Antibody-virus mixtures were added to the BHK-21 cells on ice for 20 min for synchronization of antibody-virus uptake by the cells. The chambers were moved to 37°C for 7 min. The cells were then fixed with 2% PFA, permeabilized, and stained with rabbit anti-EEA Ab. Anti-human IgG AF488 and goat anti-rabbit IgG AF568 (both from Molecular Probes) were used to detect the primary antibodies. Hoechst was used to stain nuclei. Photographs were taken at ×100 magnification with an Olympus confocal microscope.
4
Immunofluorescence Staining of SARS-CoV-2 Proteins
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Primary and secondary antibodies were diluted at 1:500 for immunofluorescence as follows: anti-Rab5 (#3547, Cell Signaling, Danvers, MA, USA), anti-Spike S1 antibody (#100715-2, BPS, San Diego, CA, USA), anti-angiotensin converting enzyme 2 (ACE2, SC390851, Santa Cruz Biotechnology, Dallas, TX, USA), anti-TOMM20 (11802-1-AP, Proteintech, Rosemont, IL, USA), anti-rabbit IgG AF488 (#A32731, Invitrogen, Waltham, MA, USA), anti-mouse IgG AF568 (#A11004, Invitrogen), and anti-human IgG AF488 (#A11013, Invitrogen). The receptor binding domain (RBD, S1) of spike protein (BT10569, R&D Systems, Minneapolis, MN, USA) and active trimer of spike protein (10586-CV, R&D Systems) were purchased from R&D Systems.
5
Antibody Binding to Plasmodium Gametes
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In vitro cultured Plasmodium falciparum NF54 gametocytes were activated to generate female gametes, which were purified with Nycodenz.26 (link) Per condition 10,000 female gametes were incubated with monoclonal antibodies diluted in SIFA buffer (1% heat-inactivated FCS, 0.05% sodium azide in PBS) for 1 hour at 4 ˚C. Samples were washed 3 times with SIFA buffer. Gametes were stained with Hoechst 33342 DNA stain (1:200 dilution) (Invitrogen, cat no. H3570) and anti-human IgG-AF488 (1:400 dilution) (Invitrogen, cat no. A-11013) for 1 hour at 4 ˚C in the dark. Gametes were then washed 3 times with SIFA buffer, fixed with 4% paraformaldehyde and imaged with an ImageXpress Pico automated cell imaging system (Molecular devices). Gametes were then analyzed with MetaXpress software (Molecular devices). Hemozoin and Hoechst-positive gametes were selected and positivity for human antibodies was determined using signal from negative control antibodies as a threshold.
6
Quantifying Female Gamete Antibody Binding
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In vitro cultured Plasmodium falciparum NF54 gametocytes were activated to generate female gametes, which were purified with Nycodenz.69 (link) Per condition 10,000 female gametes were incubated with monoclonal antibodies diluted in SIFA buffer (1% heat-inactivated FCS, 0.05% sodium azide in PBS) for 1 h at 4 ˚C. Samples were washed 3 times with SIFA buffer. Gametes were stained with Hoechst 33342 DNA stain (1:200 dilution) (Invitrogen, cat no. H3570) and anti-human IgG-AF488 (1:400 dilution) (Invitrogen, cat no. A-11013) for 1 h at 4 ˚C in the dark. Gametes were then washed 3 times with SIFA buffer, fixed with 4% paraformaldehyde and imaged with an ImageXpress Pico automated cell imaging system (Molecular devices). Gametes were then analyzed with MetaXpress software (Molecular devices). Hemozoin and Hoechst-positive gametes were selected and positivity for human antibodies was determined using signal from negative control antibodies as a threshold.
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