The cell lines used in this study were the murine lymphoma line L5178Y-R (ATCC CRL-1722), the human colorectal adenocarcinoma line HT-29 (ATCC HTB-38), the human breast cancer line MCF-7 (ATCC HTB-2), and the monkey kidney epithelial cell line MA-104 (ATCC CRL-2378.1). Peripheral blood mononuclear cells (PBMCs) were obtained from 20 mL to 30 mL (three experiments were performed) samples of blood from a healthy volunteer donor, using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Cells were maintained in RPMI-1640 medium (Life Technologies) supplemented with 10% fetal bovine serum (FBS; Life Technologies) and 1% antibiotic–antifungal solution (Life Technologies), whereas MCF-7 cells were grown in Dulbecco’s modified Eagle medium (DMEM; Life Technologies) supplemented with 10% FBS and 1% antibiotic–antifungal solution (Life Technologies) (complete culture medium). Cells were cultured at 37 °C in an atmosphere of 5% CO2.
Antibiotic antifungal solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
Antibiotic-antifungal solution is a liquid product that contains a combination of antibiotics and antifungal agents. It is designed to inhibit the growth of a wide range of microorganisms, including bacteria and fungi, in laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Ficoll paque plus, by GE Healthcare (3 mentions)
Dmem f12 medium, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Inverted phase contrast microscope, by Olympus (1 mentions)
Foetal calf serum, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Ly294002, by Abcam (1 mentions)
Ht 29, by American Type Culture Collection (1 mentions)
Mcf 7, by American Type Culture Collection (1 mentions)
Ma104, by American Type Culture Collection (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle medium dmem, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 culture medium, by Thermo Fisher Scientific (1 mentions)
Murine l5178y r lymphoma cells, by American Type Culture Collection (1 mentions)
Gw4869, by Merck Group (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Avantor (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Dmem f12 1 1 media, by Thermo Fisher Scientific (1 mentions)
10 protocols using antibiotic antifungal solution
1
Cell Culture Protocols for Diverse Cell Lines
2
Virus Isolation from Sturgeon Organs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Internal organs (spleen, kidney and heart) from each sturgeon were pooled, homogenized, diluted 1:10 in Leibovitz media supplemented with of antibiotic/antifungal solution and 10% of foetal calf serum (Invitrogen) and clarified by centrifugation. Samples were inoculated on 24-well plates with 24 h old white sturgeon skin cell line WSSK-1 (kindly provided by Prof. Ronald P. Hedrick, UC, Davis, CA, USA). These inoculated cell cultures were incubated at 20 °C for 7–10 days, with daily examination of monolayer for a cytopathic effect (CPE) [10 (link),25 (link)]. On each plate, a well with WSSK-1 cells without inoculum was used as a control to compare the differences between infected and uninfected cells. Observation to identify morphological changes of cells, such as pyknosis of nuclei and cellular fusion (syncytia), was performed according to the method [25 (link)] by using inverted phase contrast microscope (Olympus, Japan) at magnifications of 200×–400×. After first week incubation, samples with no CPE effect were re-inoculated into a new cell culture. If no CPE was observed at the end of the fourth week, the sample was considered as negative for virus isolation.
3
Ovarian Granulosa Cells Response to D-Galactose
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ovarian granulosa cells (KGN cell line; Procell) were cultured in DMEM (Sigma-Aldrich) added with 10% FBS (Sigma- Aldrich) and 1% antibiotic/antifungal solution (Invitrogen, Carlsbad, CA, USA). KGN cells in the 2nd generation were put in 6-well plates at a density of 1×105 cells/well. After 24 h, the original medium was discarded, and cells were only treated with DMEM/F12 medium (Invitrogen) for 24 h to starve the cells. Thereafter, KGN cells were assigned into 6 groups: blank (without treatment), D-gal (medium was added with 100 nM D-gal35 (link)), D-gal + EVs (medium was supplemented with 100 nM D-gal and hucMSC-EVs at a density of 1×108 particles/mL), D-gal + GW4869 (the CM was added with 100 nM D-gal and equivalent GW4869), D-gal + EVs + LY294002 (medium was supplemented with 100 nM D-gal, an equivalent volume of hucMSC-EVs, and 50 μM PI3K/Akt inhibitor LY29400236 purchased from Abcam), and D-gal + EVs + DMSO (medium was supplemented with 100 nM D-gal and equivalent volumes of hucMSC-EVs and dimethyl sulphoxide [DMSO]).
4
Murine Lymphoma and PBMC Isolation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Murine L5178Y-R lymphoma cells (ATCC CRL-1722) and PBMC were used in this study. PBMC were obtained from a 50 mL to 60 mL blood sample from a healthy donor, using Ficoll-Paque PLUS (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and following supplier’s instructions. L5178Y-R cells and PBMC were maintained in RPMI 1640 culture medium (Life Technologies, Grand Island, NY, USA), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Life Technologies) and 1% antibiotic/antifungal solution (Life Technologies) (referred as complete 1640 medium) at 37 °C in an atmosphere of 5% CO2 in air [17 (link)].
5
Murine Lymphoma and PBMC Cultivation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We used the murine lymphoma cell line L5178Y-R (ATCC CRL-1722), as well as human peripheral blood mononuclear cells (PBMC) as the control group, which were obtained from 20 to 30 mL of blood samples from healthy volunteer donors, using Ficoll–Paque Plus (GE Healthcare Life Sciences, Pittsburgh, PA) to separate white cells. Cells were maintained in RPMI-1640 medium (Life Technologies, Rockville, MD), supplemented with 10% fetal bovine serum (FBS; Life Technologies) and 1% antibiotic-antifungal solution (Life Technologies), and incubated at 37 °C in a 5% CO2 atmosphere in air.
6
Isolation and Characterization of EcESCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EcESCs, and normal human ESCs (hESCs) were isolated from the above collected tissues. The tissue samples were added with 3 times the volume of 0.25% type IV collagenase-trypsin ethylenediaminetetraacetic acid (EDTA; Sigma-Aldrich Corporation, St. Louis, MO, USA) solution and pre-warmed to 37 °C. After digestion, the tissue solutions were filtered through 150 μm and 74 μm stainless steel cell filters (GongLu; Hangzhou, China) to obtain cells, respectively. These cells were finally cultured in Dulbecco’s Modified Eagle Medium (DMEM)/F-12 (A4192001, Gibco, Waltham, CA, USA) containing 10% fetal bovine serum (FBS, 16140071, Gibco) and 1% antibiotic-antifungal solution (100 ×, 15,240,112, Gibco). After purification, the morphology was observed by immunofluorescence staining and the expression of vimentin in the cells was identified by immunofluorescence and immunohistochemistry (IHC).
For Transwell co-culture system, EcESCs, dimethyl sulfoxide (DMSO) and the sphingomyelin inhibitor GW4869 (10 μM, Sigma-Aldrich) were added to the apical chamber, and EcESCs were added to the basolateral chamber for 24 h. Cells were differently treated by DMSO or GW4869.
For Transwell co-culture system, EcESCs, dimethyl sulfoxide (DMSO) and the sphingomyelin inhibitor GW4869 (10 μM, Sigma-Aldrich) were added to the apical chamber, and EcESCs were added to the basolateral chamber for 24 h. Cells were differently treated by DMSO or GW4869.
7
Detecting Chikungunya Virus in Mosquitoes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The presence of infectious virus particles in mosquito bodies, heads, and saliva extracts were determined by focus-forming assay in C6/36 cells as previously described (21 (link)). Briefly, 96-well plates were seeded with cells, and each well was inoculated with 50 µl of saliva extract or head/body homogenate and incubated for 1 h at 28°C. Then, cells were overlaid with a 1:1 mix of carboxymethyl cellulose and Leibovitz L-15 medium supplemented with 10% FBS and 1.5× an antibiotic-antifungal solution (Gibco Life Technologies, Inc., France). After 3 days of incubation, cells were fixed for 20 min at room temperature with 3.7% formaldehyde, washed three times in phosphate-buffered saline (PBS), and incubated for 15 min with 0.5% Triton X-100 in PBS. Cells were then incubated for 1 h with a hyperimmune ascitic fluid specific to CHIKV as the primary antibody, washed three times with PBS, and incubated for 1 h at room temperature with a goat anti-mouse conjugate as the second antibody (Bio-Rad, Hercules, CA). The number of focus-forming units was determined under a fluorescence microscope. The data were analyzed qualitatively (i.e., presence or absence of infectious virus in heads/bodies) and quantitatively for saliva samples and some body and head samples.
8
Isolation and Culture of Endometrial Epithelial Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EMs ectopic endometrial epithelial cells (ecto-EECs), EMs eutopic endometrial epithelial cells (euto-EECs), and normal human endometrial epithelial cells (normal EECs) were isolated from clinically collected O-EMs tissues (within 2 h after the collection). Briefly, PBS-washed tissue samples were cut into small pieces and incubated with 0.25% type IV collagenase-trypsin EDTA (three times the volume of tissue pieces; Sigma, St. Louis, MO) at 37 °C. Following trypsinization, the tissue solution was filtered through 150 and 74 μm filters, and cells obtained were cultured in DMEM/F-12 medium supplemented with 10% FBS and 1% antibiotic-antifungal solution (Gibco, Gaithersburg, MD). All cellular experiments were performed before the fourth cell passage.
9
Adhesion Dynamics of NIH 3T3 and HEK-293 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NIH 3T3 mouse embryonic fibroblasts (ATCC CRL-1658) were cultured in DMEM/F12 (1:1) media (Gibco) with 10% FBS (VWR), 1% antibiotic/antifungal solution (Gibco) and passaged every 3 days. HEK-293 cells (Human embryonic kidney, ATCC-CRL-1573™), used as a control cell line, were cultured in DMEM, high glucose (Gibco) supplemented with 10% FBS and 1% AA.
For adhesion experiments, 3500-5000 cells were plated on functionalized surfaces in 24well plates and allowed to adhere and spread for the desired time (typically 2-4 hours) at 37 °C and 5% CO2. Adhesion assays were carried out under reduced-serum conditions (2% FBS). Following this incubation, cells were fixed for 15 min in 4% PFA or displaced by the addition of 60 µM peptide_D for the desired time (typically 1 hr) and then fixed with 4% PFA in PBS for 15 min. Cells were permeabilized in 0.2% Triton-100 for 5 min. Following permeabilization, cells were washed three times with cold PBS containing 1 mM CaCl₂ and then incubated with Alexa-fluor phalloidin (Thermo) at 1:200 in PBS for 1 hr at RT or overnight at 4 °C. Following this, cells were washed 3 times with PBS and incubated with DAPI (Sigma) at 1:1000 in PBS for 20 min. Cells were then washed 3 times in PBS and mounted on glass slides using Thermo Immu-MountImmumount. Coverslips were imaged on the GE INCell Analyzer 2200 high-content microscope.
For adhesion experiments, 3500-5000 cells were plated on functionalized surfaces in 24well plates and allowed to adhere and spread for the desired time (typically 2-4 hours) at 37 °C and 5% CO2. Adhesion assays were carried out under reduced-serum conditions (2% FBS). Following this incubation, cells were fixed for 15 min in 4% PFA or displaced by the addition of 60 µM peptide_D for the desired time (typically 1 hr) and then fixed with 4% PFA in PBS for 15 min. Cells were permeabilized in 0.2% Triton-100 for 5 min. Following permeabilization, cells were washed three times with cold PBS containing 1 mM CaCl₂ and then incubated with Alexa-fluor phalloidin (Thermo) at 1:200 in PBS for 1 hr at RT or overnight at 4 °C. Following this, cells were washed 3 times with PBS and incubated with DAPI (Sigma) at 1:1000 in PBS for 20 min. Cells were then washed 3 times in PBS and mounted on glass slides using Thermo Immu-MountImmumount. Coverslips were imaged on the GE INCell Analyzer 2200 high-content microscope.
10
Bacterial Culture and Cell Assay Techniques
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
M. smegmatis MC2155 cells obtained from ATCC, USA, were cultured on Middlebrook 7H9 media (BD Biosciences). E. coli DH5 and E. coli BL21 DE3 cells were grown in Luria–Bertani (LB) media supplemented with 30 µg/ml kanamycin. Cultures of M. smegmatis and E. coli were grown at 37 °C in a shaker incubator, set at 160 and 200 rpm, respectively. Isopropyl-β-d -1-thiogalactopyranoside (IPTG) sarcosine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), kanamycin, imidazole and staurosporine were purchased from Merck. Cell culture media and reagents, including DMEM, GlutaMAX and fetal bovine serum (FBS), and antibiotic-antifungal solution were obtained from Gibco, USA. Antibodies were obtained from BD Biosciences and Cell Signalling Technology (USA).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!