Cells detached by Accutase® (StemCell Technologies, United States), were stained for surface antigens for 30 min at 4°C with the following antibodies: APC anti-human CD206 (cat550889), PE anti-human CD80 (cat566992), FITC anti-human HLA DR (cat555560), FITC anti-human CD9 and mouse anti-human CD81 primary antibody (cat555675) (from BD Biosciences, United States), Alexa Fluor 488 goat anti-mouse IgG H&L secondary antibody (ab150117) and APC Anti-CCR5 antibody (ab176536) (from Abcam, United States). Intracellular staining was performed on fixed and permeabilized cells with Fixation/Permeabilization Kit (Cat554714, BD Biosciences, USA) according to the manufacturer’s instructions for 30 min at 4°C with mouse anti-human Cathepsin K primary antibody (ab37259) and Alexa Fluor 488 goat anti-mouse IgG H&L secondary antibody (both from Abcam, United States). The percentage of cellular death was assessed by staining with APC-conjugated annexin-V and 7-AAD, using the Annexin-V/7-AAD apoptosis detection kit (BD Biosciences, United States). As a positive control of cell death, we have exposed briefly cells to freeze-thaw cycles. Data were acquired using a FACSCanto II (Becton Dickinson, United States) and analyzed with FlowJo.v10.6.2 (Ashland, United States).
Goat anti mouse igg h l alexa fluor 488 secondary antibody
Manufactured by Abcam
Sourced in United States
Goat anti-mouse IgG H&L (Alexa Fluor 488) secondary antibody is a fluorescently labeled antibody that binds to mouse immunoglobulins. The Alexa Fluor 488 dye attached to the antibody emits green fluorescence upon excitation, allowing for the detection and visualization of mouse target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (1 mentions)
Fixation permeabilization kit, by BD (1 mentions)
Annexin v 7 aad apoptosis detection kit, by BD (1 mentions)
Accutase, by STEMCELL (1 mentions)
Apc anti human cd206, by BD (1 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Stealth rnai, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Anti 6x his tag antibody, by Abcam (1 mentions)
Ibeam smart 488, by Toptica (1 mentions)
Dmem f12, by GE Healthcare (1 mentions)
4 protocols using goat anti mouse igg h l alexa fluor 488 secondary antibody
1
Characterization of Macrophage Phenotypes
2
Investigating Multidrug Resistance Transporter
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The cell cultures media DMEM, RPMI 1640 and Opti-MEM, trypLE express, penicillin-streptomycin, L-glutamine, and transfection reagents including stealth RNAi, siRNA, and Lipofectamine RNAiMAX were from Life Technologies (Auckland, New Zealand). The apoptosis kit, myricetin, and CDCFDA (5,6-Carboxy-2’7’-Dichlorofluorescein diacetate) were from Invitrogen (Carlsbad, CA, USA). Foetal bovine serum (FBS) was from MediRay (Auckland, New Zealand). Anti-MRP2 mouse monoclonal antibody (M2 III-6) and goat anti-mouse IgG H&L (Alexa Fluor 488) secondary antibody were procured from Abcam (Cambridge, UK). The PCR kit was from Roche Diagnostics (Basel, Switzerland). Thallium and platinum standards, and MRP2 ATPase assay kit was from Sigma-Aldrich (Auckland, New Zealand). Oxaliplatin (Actavis, Auckland, New Zealand) stock solution at 5mg/ml was prepared by dissolving 100 mg powder into 20 mL milliQ water.
3
Visualizing Membrane-Bound Proteins with SIM
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SLBs were
prepared as in “Formation of SLBs ” on glass microscope coverslips (Academy, 22 × 40 mm,
0.16–0.19 mm thick). The bilayers were incubated in 2% (w/v)
BSA solution for 1 h as a blocking step and then rinsed thoroughly
with liposome buffer. The primary antibody, anti6X His Tag antibody
(Abcam) was added to the bilayers in a 1:100 dilution in 0.2% (w/v)
BSA solution, and the bilayers were incubated at 4 °C overnight.
The bilayers were washed thoroughly with liposome buffer and then
incubated in goat antimouse IgG H&L (Alexa Fluor 488) secondary
antibody (Abcam) in a 0.2% (w/v) BSA solution for 1 h. The bilayers
were washed once in liposome buffer before imaging using SIM. The
wavelengths used for excitation were 561 nm (OBIS 561, Coherent) for
the lipid bilayers and 488 nm (iBEAM-SMART-488, Toptica) for the secondary
antibody.
prepared as in “
0.16–0.19 mm thick). The bilayers were incubated in 2% (w/v)
BSA solution for 1 h as a blocking step and then rinsed thoroughly
with liposome buffer. The primary antibody, anti6X His Tag antibody
(Abcam) was added to the bilayers in a 1:100 dilution in 0.2% (w/v)
BSA solution, and the bilayers were incubated at 4 °C overnight.
The bilayers were washed thoroughly with liposome buffer and then
incubated in goat antimouse IgG H&L (Alexa Fluor 488) secondary
antibody (Abcam) in a 0.2% (w/v) BSA solution for 1 h. The bilayers
were washed once in liposome buffer before imaging using SIM. The
wavelengths used for excitation were 561 nm (OBIS 561, Coherent) for
the lipid bilayers and 488 nm (iBEAM-SMART-488, Toptica) for the secondary
antibody.
4
Isolation and Purification of Neonatal Spiral Ganglion Neurons
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Ten neonatal BALB/c mice were immersed in 75% ethanol after receiving anesthesia by inhalation of ethyl ether. The bilateral temporal bones were removed, and the spiral ganglion tissue was dissected from the stria vascularis and basilar membrane. The tissue was then digested with 0.25% trypsin (without EDTA) (cat. no. A610609; Sangon Biotech Co., Ltd., Shanghai, China) for 30 min at 37°C. After the samples were centrifuged for 8 min at 1,600 × g, the cells were resuspended and placed in 35-mm cell culture plates (2×104 cells) coated with poly-L-lysine. Cells were maintained in DMEM/F12 (GE Healthcare, Little Chalfont, UK), supplemented with 20% FBS, 10% neurotrophic factor B27 and 1% penicillin-streptomycin, and placed in an incubator with 5% CO2 and 95% air at 37°C. Cytarabine (5 µmol/ml) was used to purify the SGN for 72 h. NF-200 (1:500 dilution; cat. no. ab177487; Abcam) was the primary antibody added at 4°C for 24 h, followed by the Alexa Fluor 488 goat anti-mouse IgG (H+L) secondary antibody at 37°C for 30 min (1:500 dilution; cat. no. A11032; Thermo Fisher Scientific, Inc.) in order to identify neurons.
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