Calu-3 cells52 (link) (ATCC HTB-55) were cultured according to ATCC recommendations. All experiments were performed in these cells below passage 15. Vero E6 cells (ATCC CRL-1586; used for SARS-CoV-2 plaque assay) were grown in minimum essential medium (MEM) supplemented with 10% FBS, 1 mM sodium pyruvate and 0.1 nM non-essential amino acids, and used at passage <40. All cells were expanded in a T175 flask with 5% carbon dioxide at 37 °C. Cell density was kept between 0.25 and 2 million cells per ml. Vero E6 cells (used for TMPRSS2 proteolytic activity screening and IC50 determination) were maintained in Eagle’s minimum essential medium (EMEM) containing 10% foetal bovine serum, 2 mmol l−1l -glutamine, 100 IU ml−1 penicillin and 100 μg ml−1 streptomycin. Cm was obtained from MilliporeSigma. The SARS-CoV-2 nucleocapsid antibody (HL344) (GTX635679) was provided by Genetex; mouse anti-dsRNA antibody (J2-1904) was purchased from Scions English and Scientific Consulting33 (link); Hoechst 33342 and secondary antibodies goat anti-mouse IgG Alexa Fluor 488 (A11001) and goat anti-rabbit IgG Alexa Fluor 555 (A21428) were obtained from Invitrogen. Cell lines were screened for mycoplasma contamination using MycoAlert Mycoplasma Detection Kit (Lonza).
Goat anti mouse igg alexa fluor 488 a 11001
Manufactured by Thermo Fisher Scientific
Goat anti-mouse IgG Alexa Fluor 488 (A-11001) is a secondary antibody conjugated with the Alexa Fluor 488 fluorescent dye. It is used to detect and visualize mouse IgG primary antibodies in various immunoassay techniques.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg alexa fluor 555 a21428, by Thermo Fisher Scientific (2 mentions)
Htb 55, by American Type Culture Collection (2 mentions)
Vero e6 cell, by American Type Culture Collection (1 mentions)
Mycoalert mycoplasma detection kit, by Lonza (1 mentions)
Sterile gelatin, by Merck Group (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
40 6 diamidino 2 phenylindole dapi, by Merck Group (1 mentions)
Fv 10 asw 1, by Olympus (1 mentions)
Texas red phalloidin, by Merck Group (1 mentions)
Vectashield mounting media, by Vector Laboratories (1 mentions)
Htb 37, by American Type Culture Collection (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Cc 2540, by Lonza (1 mentions)
Hl344 gtx635679, by GeneTex (1 mentions)
Ab18199, by Abcam (1 mentions)
Dmi6000b microscope, by Leica (1 mentions)
L9393, by Merck Group (1 mentions)
Chloroquine diphosphate salt, by Merck Group (1 mentions)
Ab9465, by Abcam (1 mentions)
Ab32362, by Abcam (1 mentions)
8 protocols using goat anti mouse igg alexa fluor 488 a 11001
1
SARS-CoV-2 Infection Assay in Calu-3 and Vero E6 Cells
2
Immunofluorescence Imaging of FAK and HER2
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SKBR3 and BT-474 cells were grown on coverslips previously coated with 1% sterile gelatin (Sigma-Aldrich) and exposed for 72 h to 1-10 μg/ml Tz, 10-6M RA or the combination of both drugs. Cells were fixed with 4% paraformaldehyde for 35 min and permeabilized with 0.1% Triton for 5 min. Blocking step was performed with 0.5% bovine serum albumin solution for 30 min at room temperature. Cells were incubated with the first antibody against FAK (Mouse, clone 77, BD Biosciences) and against HER2 (Mouse, catalogue 16901-Abcam) overnight at 4°C. After washing, cells were incubated with Goat Anti-Mouse IgG-Alexa Fluor 488 (A-11001, Invitrogen) for 90 min at room temperature. The cells were washed and then stained for 35 min with Texas Red phalloidin (Sigma-Aldrich) to reveal actin and the nuclei counter stained with 40-6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) for 10 min. The coverslip cells were mounted with Vectashield mounting media (Vector Laboratories, Burlingame, CA, USA). Immunofluorescence images were captured by using a Nikon Eclipse E200 microscope (Tokyo, Japan) coupled to a high-resolution 590CU 5.0M CCD digital camera or examined under fluorescence microscopy (FV1000 Olympus Confocal Microscope) and the FV 10-ASW 1.7 software (Olympus, Japan).
3
SARS-CoV-2 Cell Culture and Antibody Evaluation
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Human Calu-3 cells (ATCC® HTB-55™) and human Caco-2 cells (ATCC® HTB-37™) were cultivated according to ATCC recommendations. All experiments were performed in these cells below passage 15. Vero E6 cells (ATCC® CRL-I1586™; used for generating SARS-CoV-2 stocks) were cultivated in MEM that was supplemented with 10% FBS, 1 mM sodium pyruvate, and 0.1 nM non-essential amino acids and used at passage <40. VeroE6/TMPRSS2 cells (JCRB 1819) were maintained in MEM supplemented with 5% FBS and penicillin and streptomycin (100 IU and 100 µg/ml, respectively). Normal human bronchial epithelial cells (NHBE), isolated from the epithelial lining of airways above the bifurcation of the lungs (NHBE-Bronchial Epi Cells; Lonza CC-2540S) were cultivated according to Lonza recommendations. The SARS-CoV-2 nucleocapsid antibody [HL344] (GTX635679) was kindly provided by GeneTex; mouse anti-dsRNA antibody (J2-1904) was purchased from Scicons English and Scientific Consulting (Long 2020); Hoechst 33,342 and secondary antibodies goat anti-mouse IgG Alexa Fluor 488 (A11001) and goat anti-rabbit IgG Alexa Fluor 555 (A21428) were obtained from Invitrogen.
4
SARS-CoV-2 Infection Assay Protocol
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Calu-3 cells (ATCC® HTB-55™) and Vero E6 cells (ATCC® CRL-158™) were cultivated according to ATCC recommendations. VeroE6/TMPRSS2 cells (Jochmans et al., 2022 (link)) were cultivated according to JCRB recommendations. Huh-7.5.1 cells were kindly provided by Dr. Francis Chisari (Scripps Research Institute)(Moradpour et al., 2004 (link)). The SARS-CoV-2 nucleocapsid antibody [HL344] (GTX635679) was kindly provided by GeneTex; mouse anti-dsRNA antibody (J2) was purchased from SCICONS English and Scientific Consulting (10010500); secondary antibodies of goat anti-mouse IgG Alexa Fluor 488 (A-11001) and goat anti-rabbit IgG Alexa Fluor 555 (A-21428) and Hoechst 33342 were obtained from Thermo Fisher Scientific. Prostratin (60857-08-1) and Gö6983 (133053-19-7) were obtained from Sigma. Bryostatin (2383), Gö6976 (2253), PEP005 (4054) and CRT 0066854 (5922) were obtained from Tocris Bioscience. N-0385 was obtained from Drs. Pierre-Luc Boudreault and Richard Leduc (Université de Sherbrooke, QC, Canada) (Shapira et al., 2022 (link)).
5
Immunohistochemical Analysis of Mouse Brain
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After transcardiac perfusion with phosphate-buffered saline and 4% paraformaldehyde (PFA), mouse brains were removed and placed in 4% PFA for 1 h and submerged in 30% sucrose overnight at 4°C. Coronal cryosections (20 µm thick) were cut from optimal cutting temperature-embedded mouse brains. For immunofluorescence labeling, brain sections were immunostained with the following primary antibodies: the endothelial marker CD31 (1:50, 550274; BD Biosciences); the ECM basement membrane component laminin (1:200, L9393; Sigma Chemical Co), MMP-9 (1:100, MAB13415, EMD Millipore, Merck KGaA), and caveolin-1 (1:1000, ab18199, Abcam). Sections were visualized with fluorophore conjugated secondary antibodies (1:300, goat anti-rat IgG-Alexa Fluor 594, A11007; goat anti-mouse IgG-Alexa Fluor 488, A11001; and goat anti-rabbit IgG-Alexa Fluor 488, A110034; ThermoFisher). Nuclear DNA dye Hoechst 33342 was used for counterstain (Gu et al., 2005 (link)). The ischemic penumbra was identified with areas of condensed nucleus changing to round, pale-stained nucleus gradually. Fluorescence images were taken with a Leica DMI 6000B microscope (Leica Microsystem). Image capture, 3D deconvolution and analysis were analyzed with LAS AF analysis tools. 3D deconvolution was used to enhance sharpness and contrast of some fluorescent images.
6
Cell Viability Assay and Protein Analysis
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MC was purchased from Tokyo Chemical Industry Co., Ltd. Quanti-MAX™ WST-8 Cell Viability Assay kit (cat. no. QM1000) and WestGlow™ FEMTO Chemiluminescent substrate (BWF0100) were obtained from Biomax Co. Ltd. ReadyShield® Protease and Phosphatase Inhibitor Cocktail, cat. no. PPC2020) and Chloroquine diphosphate salt were purchased from Sigma-Aldrich (Merck KGaA). Radioimmunoprecipitation assay (RIPA) buffer and phosphorylated (p)-STAT3 (44-384G), STAT3 (MA1-13042) glial fibrillary acidic protein (GFAP) (14-9892-82), LCN2 (PA5-79590), IP3R1 (PA1-901) and goat anti-mouse IgG Alexa Fluor 488 (A-11001) antibodies were purchased from Thermo Fisher Scientific, Inc. Actin (sc-8432), m-IgGκ BP-HRP (sc-516102) and mouse anti-rabbit IgG-HRP (cat. no. sc-2357) were purchased from Santa Cruz Biotechnology, Inc. Hematoxylin and eosin (H&E) stain kit (ab245880) was purchased from Abcam. ProLong® gold antifade reagent with DAPI mounting solution (8961) was came from Cell signaling (Danvers, MA, USA).
7
Immunofluorescence Staining of Muscle Cells
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Detailed protocols for fixation and staining of the cells have been described previously (Lewandowska et al., 2015 (link)). Briefly, medium was removed, and cells were washed with PBS. Cells were then fixed in 4% paraformaldehyde for 15 min, and washed with cold PBS. Cells were permeabilized with 0.25% Triton X-100 in PBS for 10 min and then washed. Unspecific binding was blocked by incubating cells in 1% BSA in PBS for 30 min. Cells were then incubated with primary antibodies (desmin, ab32362, Abcam; sarcomeric α-actinin, ab9465, Abcam) overnight at 4°C, washed, and then incubated with secondary antibodies (goat anti-mouse IgG AlexaFluor 488, A-11001, ThermoFisher and donkey anti-rabbit IgG AlexaFluor 555, ab150074, Abcam) for 1 h. After washing again, the cells were incubated with DAPI for 1 min, rinsed with PBS, and left in PBS at 4°C. Imaging was done using a Nikon Eclipse LVDIA-N microscope.
8
Monoclonal Antibodies Generation and Characterization
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Rat monoclonal antibodies against mouse IZUMO1 (KS64-125) and mouse SLC2A3 (KS64-10) were generated by our laboratory as described previously 44, 45 . The mouse monoclonal antibody against 1D4-tag was generated using a hybridoma cell line as a gift from Robert Molday, Ophthalmology and Visual Sciences, Centre for Macular Research, University of British Columbia, Vancouver, British Columbia, Canada 46 . Mouse monoclonal antibodies against the HA and FLAG tags were purchased from MBL (M180-3) and Sigma (F3165). The Alexa Fluor 488-conjugated Lectin PNA from Arachis hypogaea (peanut) was purchased from Thermo Fisher Scientific (L21409). The mouse monoclonal antibody against zebrafish Dcst2 was generated by the Max Perutz Labs Monoclonal Antibody Facility. Recombinant zebrafish Dcst2 (574-709), generated by VBCF Protein Technologies, served as the antigen. Horseradish peroxidase (HRP)conjugated goat anti-mouse immunoglobulins (IgGs) (115-036-062) and HRPconjugated goat anti-rat IgGs (112-035-167) were purchased from Jackson ImmunoResearch Laboratories. Fluorophore-conjugated secondary antibodies, goat antimouse IgG Alexa Fluor 488 (A11001), goat anti-mouse IgG Alexa Fluor 546 (A11018), goat anti-mouse IgG Alexa Fluor 594 (A11005), and goat anti-rat IgG Alexa Fluor 488 (A11006) were purchased from Thermo Fisher Scientific.
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