Collected cells were washed and resuspended in staining buffer (PBS 1X +1% BSA) with Fc Receptor Binding Inhibitor Antibody (Invitrogen, # 14-9161-73, Carlsbad, CA, USA) for 15 min at 4 °C. After blocking, samples were incubated in the dark for 1 h at 4 °C with a saturating concentration of anti-human primary mouse monoclonal antibodies. After two washes in PBS 1X +1% BSA, cells were incubated with secondary antibodies: Alexa Fluor 488® F(ab’)2 fragment of goat anti-mouse IgG (Invitrogen, Carlsbad, CA, USA) and/or Alexa Fluor 594® F(ab’)2 fragment of goat anti-mouse IgG (H + L) (Invitrogen). The samples were then washed with PBS 1X + 1% BSA and resuspended in 200 μL of the same buffer. The macrophages were electronically gated according to light scatter properties to exclude cell debris and contaminating lymphocytes. Fluorescence was measured using a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) and analyzed using FlowJo software (Tree Star Inc., Ashland, OH, USA).
Alexa fluor 488 f ab 2 fragment of goat anti mouse igg
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488® F(ab')2 fragment of goat anti-mouse IgG is a secondary antibody fragment conjugated to the Alexa Fluor 488 fluorescent dye. It is designed to detect and bind to mouse immunoglobulin G (IgG) antibodies.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur flow cytometer, by BD (1 mentions)
Fc receptor binding inhibitor antibody, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 f ab 2 fragment of goat anti mouse igg h l, by Thermo Fisher Scientific (1 mentions)
M200 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Huvecs, by Thermo Fisher Scientific (1 mentions)
Prestoblue cell viability kit, by Thermo Fisher Scientific (1 mentions)
Ibitreat μ slide, by Ibidi (1 mentions)
Mounting medium, by Ibidi (1 mentions)
Ix81 dsu spinning disc confocal, by Olympus (1 mentions)
Alexa fluor 594 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Ma03 be06, by BEI Resources (1 mentions)
5 protocols using alexa fluor 488 f ab 2 fragment of goat anti mouse igg
1
Immunophenotyping of Macrophages by Flow Cytometry
2
Fabrication of PDMS-based Biomimetic Membranes
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All chemicals
were purchased from Sigma-Aldrich and used without
any further purification unless stated otherwise. Poly(dimethyl silicone),
PDMS elastomer (Sylgard 184), and curing agent were obtained from
Dow Corning, USA. The secondary antibody, Alexa Fluor 488 F(ab′)2
fragment of goat antimouse IgG, HUVECs, M200 medium, LSGS, fetal bovine
serum (FBS < 5 EU/mL), and PrestoBlue cell viability kit were obtained
from Invitrogen. Fluorescent lipids 1-oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phosphocholine (NBD-PC) were purchased from
Avanti Polar Lipids, USA. All buffer solutions were prepared using
ultrapure water (Milli-Q gradient A 10 system, 18.2 MΩ/cm resistivity).
were purchased from Sigma-Aldrich and used without
any further purification unless stated otherwise. Poly(dimethyl silicone),
PDMS elastomer (Sylgard 184), and curing agent were obtained from
Dow Corning, USA. The secondary antibody, Alexa Fluor 488 F(ab′)2
fragment of goat antimouse IgG, HUVECs, M200 medium, LSGS, fetal bovine
serum (FBS < 5 EU/mL), and PrestoBlue cell viability kit were obtained
from Invitrogen. Fluorescent lipids 1-oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phosphocholine (NBD-PC) were purchased from
Avanti Polar Lipids, USA. All buffer solutions were prepared using
ultrapure water (Milli-Q gradient A 10 system, 18.2 MΩ/cm resistivity).
3
Immunofluorescence Staining of VASP Protein
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Cells were washed with PBS (twice), fixed with 4% paraformaldehyde (15 min, 37°C), washed with PBS (three times) and then permeabilized with 0.1% Triton X-100 in PBS (2 min, RT). Samples were blocked with 3% bovine serum albumin and 0.05% Tween 20 in PBS (blocking solution) for 30 min at RT, and then incubated over night at 4°C with primary antibody (anti-VASP 1:1000) diluted in blocking solution. Samples then were washed with PBS (five times) and incubated with secondary antibodies (Alexa Fluor 488 F(ab’)2 fragment of goat-anti-mouse IgG, Invitrogen), diluted (1:800) in blocking solution for 2 hours at RT. After extensive washes in PBS, cells were mounted in Ibidi mounting medium (Ibidi, Martinsried, Germany). When cells were transfected with GFP-tagged versions of VASP, transfection was performed in 8 well ibiTreat μ-Slides (Ibidi). After fixing and blocking steps, F-actin structures were stained with phalloidin (Alexa Fluor 633-Phalloidin, shown as magenta pseudocolor staining) in blocking solution. All samples were examined using an IX81 DSU Spinning Disc Confocal from Olympus with a 40x objective.
4
Antibody and Plasmid Sources for Cell Signaling Studies
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Mouse monoclonal antibodies (mAb) MA03-BE06 and IC06-BE10 [65 (link)] were obtained from BEI Resources (Manassas, VA). Alexa Fluor 488 F(ab′)2 fragment of goat anti-mouse IgG and Alexa Fluor 594 goat anti-mouse IgG were purchased from Life Technologies (Carlsbad, CA). Anti hTfR1 (CD71, Ref#555534) was obtained from BD Biosciences and isotype control (Ref#11711) were purchased from R&D systems. Anti HA (High Affinity) rat monoclonal IgG1 antibody (Cat. No. 11 867 423 001) and recombinant human IFN (Interferon-αA/D human, Cat# I4401) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Plasmid lentiCRISPR v2 was a gift from Feng Zhang (Addgene plasmid # 52961; http://n2t.net/addgene:52961 ). Lentiviral packaging plasmid pCMV-VSV-G was a gift from Bob Weinberg (Addgene plasmid # 8454; http://n2t.net/addgene:8454 ) and pLJM1-EGFP was a gift from David Sabatini (Addgene plasmid # 19319; http://n2t.net/addgene:19319 ). HA-tagged GPCs of TAMV-Ref (NC_010701.1), TAMV-FL (MK500937.1), and WWAV-AV96 (EU 123330.1) were synthetized in vitro (Genscript, Piscataway, NJ, USA). All TAMV GPs were cloned into pCAGGS plasmid, between BglII and XhoI restriction sites and HA-tagged. HA-tagged ShTfR1 was in vitro synthetized by Genscript (Piscataway, NJ, USA) and subcloned into pcDNA3.1(+) vector.
5
Antibody Characterization and Immunoblotting
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Anti TCRV NP MA03-BE06 antibody [27 (link)] was obtained from BEI Resources (Manassas, VA, USA). Rabbit antibody against ISG15 was obtained from Cell Signaling Technology (Danvers, MA, USA). Rabbit antibody against Mx1 was purchased from Proteintech (Rosemont, IL, USA). Goat antibody against CCL5 was purchased from R&D Systems (Minneapolis, MN, USA). Antibody against Vinculin (EPR8185) was obtained from Abcam (Cambridge, UK). Recombinant human IFN (interferon-αA/D human; #I4401) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor-488 F(ab′)2 fragment of goat anti-mouse IgG was obtained from Life Technologies (Carlsbad, CA, USA). Polyclonal rabbit anti-mouse and donkey anti-goat antibody conjugated with horseradish peroxidase (HRP) were obtained from Dako (Santa Clara, CA, USA).
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