Before imaging, samples were mounted in STORM imaging buffer, consisting of Tris (50 mM; pH 8.0), NaCl (10 mM), glucose (10 % wt/vol), β-mercaptoethanol (143 mM; Sigma, M3148), and an enzymatic oxygen scavenger system (1% vol/vol). The enzymatic oxygen scavenging system was added to the buffer immediately before use, and the 100× stock solution was prepared by mixing pyranose oxidase powder43 (link) (10 mg; Sigma, P4234) with catalase slurry (80 μl; Sigma, C100) in PBS (170 μl), and centrifuging the mixture at 13,000 r.p.m. for 1 min.
M3148
Manufactured by Merck Group
Sourced in United States, China, France
The M3148 is a piece of laboratory equipment manufactured by Merck Group. It serves as a precision device for various scientific applications. The core function of the M3148 is to perform accurate measurements and analysis of samples, though its specific intended use is not provided.
Automatically generated - may contain errors
Lab products found in correlation
N1250, by Solarbio (4 mentions)
Fnd500, by ExCell Bio (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Esg1107, by Merck Group (3 mentions)
Hepes, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Tryple select, by Thermo Fisher Scientific (3 mentions)
Sb431542, by Selleck Chemicals (3 mentions)
Jt4109, by Avantor (2 mentions)
P8279, by Merck Group (2 mentions)
S271 500, by Thermo Fisher Scientific (2 mentions)
M6500, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Sartorius (2 mentions)
Lif1010, by Merck Group (2 mentions)
Chir99021, by Merck Group (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
G1393, by Merck Group (2 mentions)
Doxycycline, by Merck Group (2 mentions)
L0022, by Biowest (2 mentions)
43 protocols using m3148
1
STORM Imaging Buffer Preparation
2
Labeling Biotin-Coated Microspheres with Alexa Fluor 647
3
Culture of 46C mouse embryonic stem cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
46C mESCs, kindly provided by Qi-Long Ying (University of Southern California, USA), were cultured in 0.1% gelatin-coated dishes at 37°C in 5% carbon dioxide. The medium used for routine maintenance was DMEM (Biological Industries, Israel) supplemented with 15% FBS (FND500, ExCell Bio, Australia), 1× MEM nonessential amino acids (N1250, Solarbio, China), 0.1 mM β-mercaptoethanol (M3148, Sigma) and 1000 U/ml LIF (LIF1010, Millipore, USA). Cells were digested by 0.05% Trypsin-EDTA solution every 2–3 days.
4
Western Blot Analysis of Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells grown in 6 cm dish were lysed on ice in lysis buffer (50 mM Tris-HCL [pH 7.4], 5 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM Ethylenediaminetetraacetic acid (EDTA), 1 mM Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 250 mM mannitol, 1% [v/v] Triton X-100) containing protease inhibitor complex (04693159001, Roche). The protein concentrations of the lysates were measured using the BCA Assay kit (23225, Thermo). The lysates were boiled with NuPAGE LDS-PAGE sample buffer (1771559, Invitrogen) supplemented with 5% β-mercaptoethanol (M3148, Sigma) for 5 min. Equal amounts of protein were loaded on precast NuPAGE Bis-Tris Gels (NP0321BOX, Life Technologies) followed by transfer onto nitrocellulose membrane (1620115, Bio-Rad). The immunoblotting was performed with the following antibodies: anti-PFK1 (1:1000) (ab154804, Abcam), anti-β-Actin (1:5000) (A1978, Sigma), anti-caspase 3 (1:1000) (9665s, Cell signaling), anti-cleaved-caspase 3 (1:1000) (9664s, Cell Signaling), anti-p53 (1:200) (sc-126, Santa Cruz), anti-phospho p53 (1:1000) (9284, Cell Signaling), anti-p53 (1:1000) (32532, Cell Signaling).
5
Feeder-free Culture of Mouse Embryonic Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse embryonic stem cells (mESCs) were cultured feeder-free in 0.1% gelatin-coated flasks (Sigma, G1890) under standard conditions (10% CO2, 5% O2, 90% humidity, 37 °C) in KnockOut DMEM (LifeTechnologies, 10829018), 2 mM Alanyl-glutamine (Sigma, G8541), 0.1 mM non-essential amino acids (Sigma, M7145) 15% fetal bovine serum (FBS) (Sigma, F7524), 0.1 mM β-mercaptoethanol (Sigma, M3148), ESGRO Leukemia Inhibitory Factor (LIF) (Millipore, ESG1107), Penicillin-Streptomycin (Sigma, P4333) and 2i: 1 μM MEK inhibitor PD0325901 (Sigma, PZ0162) and 3 μM GSK3 inhibitor CHIR99021 (Sigma, SML1046). Generation of H3.3 Knock-Out has been previously described6 (link). ESCs were routinely tested for mycoplasma.
6
Genetically Engineered mESC Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
E14 mouse embryonic stem cells (mESCs) were obtained from the American Type Culture Collection (ATCC). Genetically engineered mESCs, including Rif1-CKO, HA-Rif1 KI, HA-Pcgf6 KI, HA-Mga KI, and 2C::tdTomato reporter cell lines were constructed and cultured in gelatin-coated plates using high-glucose Dulbecco’s modified Eagle’s medium (DMEM, HyClone, SH3002202b) supplemented with 15% ESC-qualified fetal bovine serum (FBS, Vistech, SE200-ES), 0.1 mM 2-mercaptoethanol (Sigma, m3148), 1× non-essential amino acids (NEAA, Gibco, 11140050), and 1000 U/mL of LIF (Millipore, ESG1107). For maintenance, mESCs were cultured in the serum-free ESGRO medium (Millipore, sf001–500p). The U2OS-LacO cells (kindly provided by Professor Xuebiao Yao) and HEK293T cells were maintained in high-glucose DMEM supplemented with 10% FBS (Vistech, SE100-B). All cell lines were tested for mycoplasma contamination, and cultured at 37 °C in a humidified incubator containing 5% CO2.
7
Culturing INS-1E Cells in RPMI-1640
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
INS-1E cells were cultured in RPMI-1640 medium (10-040CV, Corning), supplemented with 11.1 mmol/l glucose (G1008.01.AG, Synth), 5% fetal bovine serum (Vitrocell, Brazil), 10 mmol/l N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES, H4034, Sigma), 100 U/ml penicillin/streptomycin (17-602F, Lonza), 1 mmol/l sodium pyruvate (P5280, Sigma), and 50 µmol/l 2-mercaptoethanol (M3148, Sigma), and kept under standard conditions (5% CO2 and 37 ºC).
8
Pancreatic β Cell Line Cultivation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mouse pancreatic β cell lines (MIN6 and β-TC6) were from ATCC. NR4A1 overexpression cell lines (designated as OV) and control cell lines (designated as NC) were generated from MIN6 cells. MKP7 knockdown cells (defined as KD-MKP7 cells) or the control cell clone (defined as CON-MKP7 cells) were generated from β-TC6 cells. MIN6 and β-TC6 cells were cultured with high glucose DMEM (CM15019/10013, M&C GENE TECHNOLOGY), supplemented with 10% Fetal Bovine Serum (A3160801, Gibco), 70 μM β-mercaptoethanol (M3148, Sigma-Aldrich), 100X penicillin-streptomycin (CC004, M&C GENE TECHNOLOGY) in a humidified environment (95% air and 5% CO2) at 37 °C. OV cells and NC cells were cultured in the same medium as MIN6 cells plus puromycin at 2 μg/ml. MKP7 knockdown cells and the control cell clone were cultured in the same medium as β-TC6 cells plus neomycin (G418) at 1 mg/ml.
9
Labeling Biotin-Coated Microspheres with Alexa Fluor 647
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A solution containing 500 μL of deionized water, 500 μL of
1×PBS, 50 μL of 9.78 μm diameter biotin-coated microsphere
solution (CP10000, Bangslab), and 0.5 μL of streptavidin-functionalized
Alexa Fluor 647 (S21374, Invitrogen) was prepared. This solution was centrifuged
for 20 min at 1340 rpm. The liquid was removed and replaced with 500 μL
of 1×PBS. 100 μL of the vortexed solution was added on the center
of a 25-mm-diameter coverslip, incubated for 20 min at RT, and sequentially
rinsed with deionized water. This coverslip was placed on a custom-made holder,
and 20 μL of imaging buffer (10% (w/v) glucose in 50 mM Tris (JT4109,
J.T.Baker), 50 mM NaCl (S271-500, Fisher Chemical), 10 mM MEA (M6500,
Sigma-Aldrich), 50 mM BME (M3148, Sigma-Aldrich), 2 mM COT (138924,
Sigma-Aldrich), 2.5 mM PCA (37580, Sigma-Aldrich), and 50 nM PCD (P8279,
Sigma-Aldrich), pH 8.0) was added on top of the coverslip. Then another cleaned
coverslip was placed on top of the imaging buffer. This coverslip sandwich was
sealed with two-component silicone dental glue.
1×PBS, 50 μL of 9.78 μm diameter biotin-coated microsphere
solution (CP10000, Bangslab), and 0.5 μL of streptavidin-functionalized
Alexa Fluor 647 (S21374, Invitrogen) was prepared. This solution was centrifuged
for 20 min at 1340 rpm. The liquid was removed and replaced with 500 μL
of 1×PBS. 100 μL of the vortexed solution was added on the center
of a 25-mm-diameter coverslip, incubated for 20 min at RT, and sequentially
rinsed with deionized water. This coverslip was placed on a custom-made holder,
and 20 μL of imaging buffer (10% (w/v) glucose in 50 mM Tris (JT4109,
J.T.Baker), 50 mM NaCl (S271-500, Fisher Chemical), 10 mM MEA (M6500,
Sigma-Aldrich), 50 mM BME (M3148, Sigma-Aldrich), 2 mM COT (138924,
Sigma-Aldrich), 2.5 mM PCA (37580, Sigma-Aldrich), and 50 nM PCD (P8279,
Sigma-Aldrich), pH 8.0) was added on top of the coverslip. Then another cleaned
coverslip was placed on top of the imaging buffer. This coverslip sandwich was
sealed with two-component silicone dental glue.
10
Culture of Mouse ESCs and Human iPSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 46C mouse ESCs were kindly provided by Qi-Long Ying (The University of Southern California) and were cultured in 0.1% gelatin-coated dishes at 37 °C in 5% CO2. The conventional cell culture conditions were Dulbecco's modified Eagle's medium (Biological Industries) supplemented with 10% fetal bovine serum (FND500, ExCell Bio), 1× MEM nonessential amino acids (N1250, Solarbio), 1× penicillin/streptomycin (P1400, Solarbio), 0.1 mM β-mercaptoethanol (M3148, Sigma), and LIF (made in house). Human transgene-free iPSCs were kindly provided by Nuwacell Ltd (ZSSY-001) and were cultured in ncTarget (RP01020, Nuwacell Ltd). Cells were dissociated using EDTA solution (RP01007, Nuwacell Ltd) every 3 to 5 days.
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!