The GCs were isolated from antral follicles of the ovary as per the method described by Campbell et al. (1996) with minor modifications. Antral follicles (~3.5mm) were punctured by 26G needle (Becton Dickenson) and the follicular fluid was aspirated gently and transferred into 1.5 ml Eppendorf tube containing McCoy's 5A media (Sigma). Filtered media was added to the above fluid to make the volume of 1ml and the tube was stood for 10 min at 37°C to settle down the theca cells. The supernatant was gently aspirated in a new 1.5 mL Eppendorf tube and the content was briefly centrifuged at 300 g for 7 min. The pellet of GCs was then suspended and cultured in 500 µL McCoy's 5A media containing NaHCO 3 2.2g/L and HEPES 4.76g/L supplemented with 2mM L-glutamine, 0.1% BSA, 2 μg/ mL insulin, 1 μg/mL FSH, 20 ng/mL IGF-I, 0.02 IU/mL LH, 0.8 μg/mL selenium with 5% (v/v) FBS (all purchased from Sigma) in 4-well plate. After 6-7 days of primary culture, GCs were trypsinized by 0.25% Trypsin-EDTA (Sigma) and sub-cultured in 24-well plate. The GCs were cultured in McCoy's 5A media at 37°C in 5% CO 2 for further study.
Mccoy s 5a media
Manufactured by Merck Group
Sourced in United States
McCoy's 5A media is a cell culture medium used to support the growth and maintenance of various cell types in vitro. It is a balanced salt solution that provides the necessary nutrients, vitamins, and other components required for cell proliferation and survival.
Automatically generated - may contain errors
Lab products found in correlation
Spectramax plus 384 reader, by Molecular Devices (20 mentions)
Penicillin streptomycin solution, by Merck Group (20 mentions)
Mts reagent, by Promega (20 mentions)
L glumtamine, by Merck Group (17 mentions)
L glutamine, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Campylobacter selective supplement skirrow, by Thermo Fisher Scientific (2 mentions)
Mueller hinton broth (mhb), by Thermo Fisher Scientific (2 mentions)
Campygen gas pack, by Thermo Fisher Scientific (2 mentions)
25 cm2 tissue culture flask, by Corning (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Glutamax, by Thermo Fisher Scientific (2 mentions)
Anaerocult a system, by Merck Group (1 mentions)
L cysteine, by Merck Group (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Nucblue live readyprobes reagent, by Thermo Fisher Scientific (1 mentions)
Sk br 3, by Merck Group (1 mentions)
37 protocols using mccoy s 5a media
1
Isolation and Culture of Granulosa Cells
2
Cultivation and Maintenance of Gut Bacteria
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Bacterial cultures were maintained as previously described [17 (link),18 (link)]. B. infantis was stored in de Man–Rogosa–Sharpe (MRS) (Difco, Sparks, MD, USA) broth containing 50% glycerol at −80 °C. The strain was cultured twice in MRS media supplemented with L-cysteine (0.05% w/v) (Merck, Dannstadt, Germany) prior to use, and was routinely grown overnight at 37 °C under anaerobic conditions generated using an Anaerocult A system (Merck, Dannstadt, Germany).
C. jejuni 81–176 was stored in Mueller Hinton broth (Oxoid, Ireland c/o Fannin Healthcare, Dublin, Ireland) containing 50% glycerol at −80 °C and cultured directly from storage onto Mueller-Hinton agar plates. The pathogen was grown under microaerophilic conditions generated using CampyGen gas packs (Oxoid), for 48 h at 37 °C as previously described [19 (link),22 (link)]. Prior to pathogen inhibition assays, C. jejuni 81–176 was grown on Mueller-Hinton agar and then transferred to biphasic media in 25 cm2 tissue culture flasks (Corning, New York, NY, USA) consisting of Mueller-Hinton agar supplemented with Campylobacter selective supplement (Skirrow), (Oxoid) and 6 mL of McCoy’s 5A media (Merck) supplemented with 2% fetal bovine serum (FBS). The flask was then incubated for 24 h under microaerophilic conditions at 37 °C.
C. jejuni 81–176 was stored in Mueller Hinton broth (Oxoid, Ireland c/o Fannin Healthcare, Dublin, Ireland) containing 50% glycerol at −80 °C and cultured directly from storage onto Mueller-Hinton agar plates. The pathogen was grown under microaerophilic conditions generated using CampyGen gas packs (Oxoid), for 48 h at 37 °C as previously described [19 (link),22 (link)]. Prior to pathogen inhibition assays, C. jejuni 81–176 was grown on Mueller-Hinton agar and then transferred to biphasic media in 25 cm2 tissue culture flasks (Corning, New York, NY, USA) consisting of Mueller-Hinton agar supplemented with Campylobacter selective supplement (Skirrow), (Oxoid) and 6 mL of McCoy’s 5A media (Merck) supplemented with 2% fetal bovine serum (FBS). The flask was then incubated for 24 h under microaerophilic conditions at 37 °C.
3
Cultivation of Campylobacter jejuni 81-176
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Campylobacter jejuni 81–176 (C. jejuni) is a well-characterized, mobile flagellated invasive strain which has been used in many previous studies [17 (link),18 (link)]. The pathogen was stored in Mueller–Hinton broth (Oxoid, Ireland c/o Fannin Healthcare, Dublin, Ireland) containing 50% glycerol at −80 °C and cultured directly from storage onto Mueller–Hinton agar plates. The pathogen was grown under microaerophilic conditions generated using CampyGen gas packs (Oxoid), for 48 h at 37 °C. Prior to pathogen inhibition assays, C. jejuni 81–176 was grown on Mueller–Hinton agar and then transferred to biphasic media in 25 cm2 tissue culture flasks (Corning, NY, USA) consisting of Mueller–Hinton agar supplemented with Campylobacter selective supplement (Skirrow), (Oxoid) and 6 mL of McCoy′s 5A media (Merck) supplemented with 2% FBS. The flask was then incubated for 24 h under microaerophilic conditions at 37 °C.
4
Establishing Doxorubicin-Resistant Cell Lines
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HCT116 and DLD1 human cells were bought from ATCC. These cells were cultured in McCoy's 5A media (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) at 37°C in 5% CO2. Doxorubicin‐resistant HCT116 DR cells were established as previously described (Choi, Kim, Choi, Kim, & Lee, 1996 ).
5
HCT116 Cell Viability Screening
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Example 5
Compounds can be screened for single agent activity against HCT116 colorectal cancer cells using a 96 h cell viability (MTS) assay. HCT116 are plated at 470 cells per well in 96-well polystyrene plates (Costar 3596) in 150 μl of McCoy's 5A media (Sigma M8403) supplemented with 10% foetal bovine serum (JRH Biosciences 12003), Penicillin/Streptomycin solution diluted 1:100 (Sigma P7539), and 2 mM L-glumtamine (Sigma G7513), and allowed to adhere overnight at 37° C. in 5% CO2. Compounds are then added to the cell media in 2-fold serial dilutions from a top final concentration of 10 μM as a full matrix of concentrations in a final cell volume of 200 μl, and the cells are then incubated at 37° C. in 5% CO2. After 96 h, 40 μl of MTS reagent (Promega G358a) is added to each well and the cells are incubated for 1 h at 37° C. in 5% CO2. Finally, absorbance is measured at 490 nm using a SpectraMax Plus 384 reader (Molecular Devices) and IC50 values can be calculated.
6
HCT116 Colorectal Cancer Cell Viability Assay
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Example 18
Compounds can be screened for single agent activity against HCT116 colorectal cancer cells using a 96h cell viability (MTS) assay. HCT116 are plated at 470 cells per well in 96-well polystyrene plates (Costar 3596) in 150l of McCoy's 5A media (Sigma M8403) supplemented with 10% foetal bovine serum (JRH Biosciences 12003), Penicillin/Streptomycin solution diluted 1:100 (Sigma P7539), and 2 mM L-glumtamine (Sigma G7513), and allowed to adhere overnight at 37° C. in 5% CO2. Compounds are then added to the cell media in 2-fold serial dilutions from a top final concentration of 10 μM as a full matrix of concentrations in a final cell volume of 200l, and the cells are then incubated at 37° C. in 5% CO2. After 96 h, 40 μl of MTS reagent (Promega G358a) is added to each well and the cells are incubated for 1 hr. at 37° C. in 5% CO2. Finally, absorbance is measured at 490 nm using a SpectraMax Plus 384 reader (Molecular Devices) and IC50 values can be calculated.
7
Screening Compounds for Colorectal Cancer Cells
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Example 22
Compounds can be screened for single agent activity against HCT116 colorectal cancer cells using a 96 h cell viability (MTS) assay. HCT116 are plated at 470 cells per well in 96-well polystyrene plates (Costar 3596) in 150 μl of McCoy's 5A media (Sigma M8403) supplemented with 10% foetal bovine serum (JR H Biosciences 12003), Penicillin/Streptomycin solution diluted 1:100 (Sigma P7539), and 2 mM L-glumtamine (Sigma G7513), and allowed to adhere overnight at 37° C. in 5% CO2. Compounds are then added to the cell media in 2-fold serial dilutions from a top final concentration of 10 μM as a full matrix of concentrations in a final cell volume of 200μ1, and the cells are then incubated at 37° C. in 5% CO2. After 96 h, 40 μl of MTS reagent (Promega G358a) is added to each well and the cells are incubated for 1 h at 37° C. in 5% CO2. Finally, absorbance is measured at 490 nm using a SpectraMax Plus 384 reader (Molecular Devices) and IC50 values can be calculated.
8
Monitoring Mitosis in Synchronized U2OS Cells
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Stable U2OS cells expressing A1aY1 with an N-terminal TagRFP-T (red tyrosination sensor) were grown up to 50% confluency in McCoy’s 5A media (Sigma-Aldrich; M4892) in 37°C in a 5% CO2 incubator. Cells were arrested at S phase of the cell cycle with 2.5 mM thymidine for 16–20 h at 37°C and released for cell cycle progression for 8–9 h in fresh media. Similarly, a second S-phase arrest in 2.5 mM thymidine was performed for 20–24 h and released for 8 h in fresh media, followed by 20 ng/ml nocodazole (Sigma-Aldrich; M1404) treatment for 4 h. The synchronized cells (30–40%) were imaged for mitosis after staining the nucleus of the cells with DAPI (1 μg/ml) for 15 min or with NucBlue live ready probes reagent (Invitrogen; catalog no. R37605).
9
Screening Compounds for Colorectal Cancer
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Example 18
Compounds can be screened for single agent activity against HCT116 colorectal cancer cells using a 96 h cell viability (MTS) assay. HCT116 are plated at 470 cells per well in 96-well polystyrene plates (Costar 3596) in 150 μl of McCoy's 5A media (Sigma M8403) supplemented with 10% foetal bovine serum (JRH Biosciences 12003), Penicillin/Streptomycin solution diluted 1:100 (Sigma P7539), and 2 mM L-glumtamine (Sigma G7513), and allowed to adhere overnight at 37° C. in 5% CO2. Compounds are then added to the cell media in 2-fold serial dilutions from a top final concentration of 10 μM as a full matrix of concentrations in a final cell volume of 200 μl, and the cells are then incubated at 37° C. in 5% CO2. After 96 h, 40 μl of MTS reagent (Promega G358a) is added to each well and the cells are incubated for 1 hr. at 37° C. in 5% CO2. Finally, absorbance is measured at 490 nm using a SpectraMax Plus 384 reader (Molecular Devices) and IC50 values can be calculated.
10
Compound Screening for HCT116 Colorectal Cancer
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Example 8
Compounds can be screened for single agent activity against HCT116 colorectal cancer cells using a 96 h cell viability (MTS) assay. HCT116 are plated at 470 cells per well in 96-well polystyrene plates (Costar 3596) in 150 μl of McCoy's 5A media (Sigma M8403) supplemented with 10% foetal bovine serum (JRH Biosciences 12003), Penicillin/Streptomycin solution diluted 1:100 (Sigma P7539), and 2 mM L-glumtamine (Sigma G7513), and allowed to adhere overnight at 37° C. in 5% CO2. Compounds are then added to the cell media in 2-fold serial dilutions from a top final concentration of 10 μM as a full matrix of concentrations in a final cell volume of 200 μl, and the cells are then incubated at 37° C. in 5% CO2. After 96 h, 40 μl of MTS reagent (Promega G358a) is added to each well and the cells are incubated for 1 h at 37° C. in 5% CO2. Finally, absorbance is measured at 490 nm using a SpectraMax Plus 384 reader (Molecular Devices) and IC50 values can be calculated.
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