The 3T3-L1 organoids were generated by a hanging droplet spheroid three dimension (3D) culture system as described recently28 (link). Briefly, to facilitate stable morphology, methylcellulose (Methocel A4M) was added to the growth medium. Prior to seeding the hanging drop culture plate (# HDP1385, Sigma-Aldrich), cells were cultured in 100 mm or 150 mm dishes until reaching approximately 90% confluence. After washing with a phosphate buffered saline (PBS), cells were detached using 0.25% Trypsin/EDTA and resuspended in growth medium. After centrifugation for 5 min at 300 g, the cell pellet was re-suspended in a growth medium containing 0.25% w/v Methocel A4M. Volume was adjusted so that 20,000 cells were contained in the 28 μL solution, and 28 μL drops were placed into each well of the drop culture plate (defined as 3D/Day 0). An organoid medium (i.e. growth medium with 0.25% w/v Methocel A4M) was used throughout the duration of the spheroid culture. On every following day, 14 μL of the culture medium was removed and a fresh 14 μL culture medium was added to each well.
Pantothenate, Calcium
It plays a crucial role in the body's ability to convert food into cellular energy.
Calcium pantothenate is commonly used as a dietary supplement to support overall health and wellness.
This compound can be found naturally in a variety of foods, including meat, dairy, legumes, and whole grains.
Reasearchers continue to explore the potential benefits of Calcium Pantothenate supplementatioin for conditions such as stress, fatigue, and immune function.
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The 3T3-L1 organoids were generated by a hanging droplet spheroid three dimension (3D) culture system as described recently28 (link). Briefly, to facilitate stable morphology, methylcellulose (Methocel A4M) was added to the growth medium. Prior to seeding the hanging drop culture plate (# HDP1385, Sigma-Aldrich), cells were cultured in 100 mm or 150 mm dishes until reaching approximately 90% confluence. After washing with a phosphate buffered saline (PBS), cells were detached using 0.25% Trypsin/EDTA and resuspended in growth medium. After centrifugation for 5 min at 300 g, the cell pellet was re-suspended in a growth medium containing 0.25% w/v Methocel A4M. Volume was adjusted so that 20,000 cells were contained in the 28 μL solution, and 28 μL drops were placed into each well of the drop culture plate (defined as 3D/Day 0). An organoid medium (i.e. growth medium with 0.25% w/v Methocel A4M) was used throughout the duration of the spheroid culture. On every following day, 14 μL of the culture medium was removed and a fresh 14 μL culture medium was added to each well.
Experiments were started by initially growing cultures in 300mL of the appropriate defined media in batch phase. Once the cultures reached saturation, chemostat flow was initiated. Cultures were grown at a dilution rate of 0.17 volumes/hour. Daily samples were taken from the overflow in order to determine optical density at 600 nm, cell count and viability; perform microscopy; and make archival glycerol stocks. We confirmed that all evolved haploid clones maintained the same mating type as the founder by backcrossing the evolved strain to the isogenic ancestral strain of the opposite mating type. Clones from three of the twelve evolved diploid populations exhibited reduced sporulation efficiency, but did not mate inappropriately.
To generate 3D spheroids under culture conditions identical to those used for the 2D cell culture except culture plates, a hanging droplet spheroid 3D culture system, described recently [6 (link),14 (link)], was used. Briefly, 3T3-L1 cells were cultured in 100 mm or 150 mm dishes as described above. After reaching approximately 90% confluence, the cells were detached by treatment with 0.25% trypsin/EDTA after washing with phosphate-buffered saline (PBS) and then resuspended in the growth medium. These cell suspensions were divided into conventional 2D cultures and 3D spheroid cultures. The 2D-cultured 3T3-L1 cells were again maintained 100 mm 2D culture dishes until Day 7, with medium changes daily. Alternatively, a cell suspension containing approximately 20,000 cells in a 28 μL culture medium was then placed into each well of the 3D drop culture plate (# HDP1385, Sigma-Aldrich, St. Louis, MO, USA). This timing was defined as 3D/Day 0, and 14 μL of the culture medium was then replaced with 14 μL of fresh culture medium in each well daily until Day 7 [7 (link),8 (link),10 (link)]. On Day 7, both 2D- and 3D-cultured 3T3-L1 cells were each collected and further processed for use in the RNA-sequencing analysis, as described below.
To obtain 3T3-L1 organoids, 3T3-L1 preadipocytes were grown in 3D pre-culture medium (HG-DMEM containing 8 mg/L d-biotin, 4 mg/L calcium pantothenate, 100 U/mL penicillin, 100 μg/mL streptomycin and 10% CS) in 150 mm dish as above. When the cultures reached approximately a 90% confluence, the cells were washed with phosphate buffered saline (PBS), detached using 0.25% Trypsin/EDTA and resuspended in 3D pre-culture medium containing 0.25% w/v Methocel A4M (3D organoid medium). Approximately 20,000 cells in the 28μL 3D organoid medium were placed into each well of the drop culture plate (defined as 3D/Day 0). On alternate days, 14 μL of the medium was substituted with 14 μL of fresh medium in each well.
For the induction of adipogenic differentiation, two days after the 2D cells in the 2D culture medium or 3D organoids at Day 1 in the 3D organoid medium reached confluence, there were processed by supplementation with 250 nM dexamethasone, 10 nM T3, 10 μM troglitazone, and 1 μg/ml insulin during the initial two days, and during the following 4 days with 10 μM troglitazone and 1 μg/ml insulin. To study the efficacy of several drugs, 100 nM prostaglandin F2α (PGF2α), 100 nM omidenepag (OMD), or 100 nM butaprost (Buta) were added during their adipogenic differentiation. In terms of their corresponding receptor, PF and EP2 receptors, of these drugs, we confirmed their positive expressions with the 2D and 3D cultured 3T3-L1 cells (Supplemental Fig.
Phase contrast images of the 3D organoids were captured in a × 4 objective lens using an inverted microscope (Nikon ECLIPSE TS2; Tokyo, Japan). For measurement of each organoid size, the largest cross-sectional area (CSA) was calculated using the Image-J software version 1.51n (National Institutes of Health, Bethesda, MD).
S. pneumoniae was grown in C+Y medium at 37°C. C+Y was adapted from Adams and Roe (Adams and Roe 1945 (link)) and contained the following compounds: adenosine (68.2 μM), uridine (74.6 μM), L-asparagine (302 μM), L-cysteine (84.6 μM), L-glutamine (137 μM), L-tryptophan (26.8 μM), casein hydrolysate (4.56 g L-1), BSA (729 mg L-1), biotin (2.24 μM), nicotinic acid (4.44 μM), pyridoxine (3.10 μM), calcium pantothenate (4.59 μM), thiamin (1.73 μM), riboflavin (0.678 μM), choline (43.7 μM), CaCl2 (103 μM), K2HPO4 (44.5 mM), MgCl2 (2.24 mM), FeSO4 (1.64 μM), CuSO4 (1.82 μM), ZnSO4 (1.58 μM), MnCl2 (1.29 μM), glucose (10.1 mM), sodium pyruvate (2.48 mM), saccharose (861 μM), sodium acetate (22.2 mM) and yeast extract (2.28 g L-1).
We can control competence development by changing the pH in the medium. The underlying mechanism it is not fully understood, but it is believed that is related to the production and export of CSP (Moreno-Gámez et al., 2017 (link)). For this reason, we always grow a preculture in C+Y at pH 6.8, because at this pH, even the hypercompetent strains such as ΔlytB or Δpbp3 mutants, are not able to accumulate enough CSP to induce competence before cells reach stationary phase.
Most recents protocols related to «Pantothenate, Calcium»
Example 2
100 mg of the Sarcodon aspratus extracts according to the present invention;
an appropriate amount of a vitamin mixture;
70 μg of vitamin A acetate;
1.0 mg of vitamin E;
0.13 mg of vitamin B1;
0.15 mg of vitamin B2;
0.5 mg of vitamin B6;
0.2 μg of vitamin B12;
10 mg of vitamin C;
10 μg of biotin;
1.7 mg of nicotinic acid amide;
50 μg of folate;
0.5 mg of calcium pantothenate;
an appropriate amount of a mineral mixture;
1.75 mg of ferrous sulfide;
0.82 mg of zinc oxide;
25.3 mg of magnesium carbonate;
15 mg of potassium phosphate monobasic;
55 mg of dicalcium phosphate;
90 mg of potassium citrate;
100 mg of calcium carbonate; and
24.8 mg of magnesium chloride.
The composition ratio of the vitamins and the mineral mixture described above may be determined according to a composition ratio used in general functional health foods, and the combination ratio of the vitamins and the mineral mixture may be arbitrarily determined. According to a conventional method of preparing functional health foods, these components are mixed, granules are prepared, and the granules are used to prepare a composition for a functional health food.
Example 3
Clostridium histolyticum ATCC 21000, strain 004 was inoculated into the starting culture with M #1 or M #2 and incubated at 37° C. for 16 hours. Ten milliliters of the starting culture (M #1 or M #2) and 10 mL Mg/vitamin solution (prepared separately by dissolving 8 g MgSO4, 1.2 g ferrous sulfate, 0.05 g riboflavin, 0.1 g Niacin, 0.1 g Calcium pantothenate, 0.1 g pimelic acid, 0.1 g pyridoxine, and 0.1 g thiamine in 1100 mL water, followed by sterilization by 0.22 μm filtration) was then transferred to each liter of M #3 or M #4 (or a variation thereof), and incubated for 22 hours. Clostridium histolyticum grew well with the OD600 reaching >2.5.
Example 3
Clostridium histolyticum ATCC 21000, strain 004 was inoculated into the starting culture with M #1 or M #2 and incubated at 37° C. for 16 hours. Ten milliliters of the starting culture (M #1 or M #2) and 10 mL Mg/vitamin solution (prepared separately by dissolving 8 g MgSO4, 1.2 g ferrous sulfate, 0.05 g riboflavin, 0.1 g Niacin, 0.1 g Calcium pantothenate, 0.1 g pimelic acid, 0.1 g pyridoxine, and 0.1 g thiamine in 1100 mL water, followed by sterilization by 0.22 pm filtration) was then transferred to each liter of M #3 or M #4 (or a variation thereof), and incubated for 22 hours. Clostridium histolyticum grew well with the OD600 reaching >2.5.
EHP Labs OxyShred thermogenic fat burner ingredients list
OxyShred (one serving) | Amount/serving | % DV |
---|---|---|
Calories | 5 | |
Total carbohydrate | 1.0 g | <1 |
Dietary fiber | 0.2 g | 4* |
Vitamin C | 173 mg | 193 |
Thiamin | 0.56 mg | 46 |
Riboflavin | 0.78 mg | 60 |
Niacin | 20 mg | 123 |
Vitamin B6 | 0.98 mg | 58 |
Vitamin B12 | 0.9 mcg | 38 |
Pantothenic acid | 1.7 mg | 34 |
Chromium picolinate | 10 mcg | 3 |
Fat burning matrix | 2003 mg | |
Immunity booster & prebiotic complex | 625 mg | |
Mood enhancer matrix | 851 mg | |
Full B vitamin spectrum | 24.59 mg |
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More about "Pantothenate, Calcium"
Tween 80, a non-ionic surfactant, can be used alongside Calcium Pantothenate for improved solubility and bioavailability.
Nicotinic Acid, or Vitamin B3, and L-Leucine are other B-complex vitamins that work synergistically with Calcium Pantothenate to support energy levels and overall health.
Biotin, also known as Vitamin B7, is another important cofactor in metabolic processes.
Solvents like Acetonitrile and DMSO can be utilized for Calcium Pantothenate extraction and analysis, while Dexamethasone, a synthetic glucocorticoid, may interact with Calcium Pantothenate supplementation.
Optima LC-MS grade methanol provides high purity for liquid chromatography-mass spectrometry quantification of Calcium Pantothenate.
Whether you're studying the biochemistry, pharmacology, or nutritional aspects of this essential nutrient, PubCompare.ai can help you find the best protocols and optimize your Pantothenate, Calcium research.