The Mycobacterium haemophilum type strain (DSM 44634; ATCC 29548) was cultured at 30°C in Middlebrook 7H9 broth supplemented with 0.2% glycerol, 10% OADC enrichment (Becton, Dickinson), 0.05% tyloxapol or Tween 80, and 100 µM hemin (bovine [Sigma]; 7H9-hemin) or as described above. Comparative growth studies utilized mc26230, a ΔRD1 ΔpanCD derivative of M. tuberculosis H37Rv which is approved for use in BSL2 facilities at the Albert Einstein College of Medicine (31 (link)), and an mbtB deletion mutant in the mc26230 background (see Text S1 in the supplemental material for further details on our materials and methods). Media for growth of mc26230 and mc26230 ΔmbtB were supplemented with 24 µg/ml d -pantothenate (Sigma).
D pantothenate
Manufactured by Merck Group
Sourced in Portugal
D-pantothenate is a form of pantothenic acid, also known as vitamin B5. It is an essential nutrient that serves as a precursor for the synthesis of coenzyme A, a vital cofactor involved in numerous metabolic processes within the body.
Automatically generated - may contain errors
Lab products found in correlation
Dexamethasone (dex), by Merck Group (3 mentions)
3 isobutyl 1 methylxanthine ibmx, by Merck Group (2 mentions)
Insulin, by Merck Group (2 mentions)
Troglitazone, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Glutamax 1, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Harvard Bioscience (2 mentions)
Streptavidin microbeads, by Miltenyi Biotec (2 mentions)
Hemin, by Merck Group (1 mentions)
Oadc enrichment, by BD (1 mentions)
D biotin, by Merck Group (1 mentions)
Rosiglitazone, by Enzo Life Sciences (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
H insulin, by Merck Group (1 mentions)
Isobutylmethylxanthine ibmx, by Merck Group (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (1 mentions)
Dmem f12 media, by Thermo Fisher Scientific (1 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (1 mentions)
6 well plate, by Corning (1 mentions)
7 protocols using d pantothenate
1
Culturing Mycobacterium haemophilum and Mutants
2
Adipogenic Differentiation of hMADS Cells
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Human multipotent abdominal subcutaneous adipose-derived mesenchymal stem cells (hMADS) were obtained from overweight/obese men with impaired glucose metabolisms and pooled and differentiated into the adipogenic lineage. Therefore, cells were seeded at a density of 2000 cells∙cm−2 and cultured, as described previously [27 ,28 (link)]. Human multipotent adipose-derived mesenchymal stem cells (hMADS) were kept in a proliferation medium containing DMEM and Ham's F-12 (DMEM-Ham's F-12) Nutrient Mixture (no. 31330-095, Gibco), 10 % FBS (Bodinco BV), and 1x Antibiotic-Antimycotic (Gibco). At ~80 % confluence, a differentiation medium was added to the cells containing DMEM-Ham's F-12, 3 % FBS (Bodinco BV), 1x Antibiotic-Antimycotic (Gibco), 33 μM D-Biotin (no. B4693, Sigma), 17 μM d -pantothenate (no. P5155, Sigma), 0.1 μM h-insulin (no. 91077C, Sigma), 1 μM dexamethasone (no. D4902, Sigma), 250 μM 3-isobutyl-1-methylxanthine (IBMX, no. I5879, Sigma), and 5 μM rosiglitazone (no. ALX-350-125-M025, Enzo Life Sciences). After 7 days, IBMX and rosiglitazone were removed from the medium. All cells were proliferated and differentiated under 21 % O2, and thereafter, exposed to either 10 % O2 continuously (resembling physiological normoxia), or to MIH consisting of 3 × 2 h cycles per day, alternating between 5 and 10 % O2, during the final 7 days of cell culture.
3
Adipogenic Differentiation of hASCs in Gellan Gum
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Adipogenic differentiation of hASCs within gellan gum hydrogel particles was induced as previously described [24 ] with some modifications. After culturing cell-laden hydrogel particles in α-MEM medium for three days, medium was changed to an adipogenic induction medium (IM) consisting of α-MEM supplemented with 10% FBS, 1% ATB, 34 µM of D-pantothenate (Sigma-Aldrich, Sintra, Portugal) and 66 µM of biotin (Sigma-Aldrich, Sintra, Portugal), 200 nM of insulin (Sigma-Aldrich, Sintra, Portugal), 1 µM of dexamethasone (Sigma-Aldrich, Sintra, Portugal), 250 µM of 3-isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich, Sintra, Portugal), and 5 µM of troglitazone (Sigma-Aldrich, Sintra, Portugal). Cells were cultured for further 3, 6, 15 and 21 days. An additional condition was set after days six of induction, by changing the medium to maintenance medium (MM)—IM without IBMX and troglitazone—and culture the cell-laden hydrogel particles for a further nine days.
4
Adipogenic and Osteogenic Induction of pASCs
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pASCs (n = 4) were seeded in duplicate at the concentration of 5.0 × 104 into 24-well plates (p = 1) and cultured until they reached 60–70% confluency. Adipogenic differentiation was induced according to the procedure described previously by Yu et al.58 (link). Briefly, adipogenic medium I consisted of DMEM/F12 supplemented with 5% FBS, 66 μM biotin (Sigma-Aldrich Co.), 34 μM d-pantothenate (Sigma-Aldrich Co.), 200 nM insulin (Sigma-Aldrich Co.), 1 μM dexamethasone (Sigma-Aldrich Co.), 250 μM isobutyl-methylxanthine (IBMX, Sigma-Aldrich Co.), and 5 μM troglitazone (Sigma-Aldrich Co.). Adipogenic medium II consisted of DMEM/F12 supplemented with 5% FBS, 66 μM biotin, 34 μM d-pantothenate, 200 nM insulin, and 1 μM dexamethasone. The pASCs were incubated for 3 days in adipogenic medium I, and for the next 11 days, cells were cultured in adipogenic medium II at 39 °C in 21% O2 and 5% humidified CO2. For osteogenic induction, Stem-Pro1 Osteogenesis Differentiation Kits (Life Technologies by Thermo Fisher Scientific) were used according to the manufacturer’s instructions.
5
Isolation and Culture of Urothelial Carcinoma Cells
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Urothelial carcinoma tumors from four patients were obtained from the University Hospital Bonn under the ethical approval number 363/20. Tumors were minced and digested as described above for the murine adipose tissue. Cells were washed in 2 mL MACS buffer (0.5% BSA and 2 mmol/L EDTA in PBS) and labeled with biotin-conjugated antibodies against lineage markers of endothelial cells (anti-CD31; clone AC128; #130–119–893), immune cells (anti-CD45; clone 5B1; #130–113–116), and epithelial cells [anti-EpCAM (CD326); clone REA764; #130–110–997]. Cells were then incubated with Streptavidin MicroBeads (Miltenyi, #130–048–101) and subjected to MACS. The lineage depleted cells were harvested and maintained in DMEM/F12, supplemented with 1% GlutaMAX-I, 1% penicillin–streptomycin (all Gibco/Life Technologies), 10% FCS (Biochrom), 33 mmol/L biotin (Sigma), and 17 mmol/L D-pantothenate (Sigma) at 37°C with 5% CO2. Conditioned media (CM) was collected when cells reached confluence of approximately 70%. All antibodies were purchased from BioLegend.
6
Differentiation of Human SGBS Pre-adipocytes
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Human SGBS pre-adipocytes of passage 7 were cultured in Gibco™ Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) Media (Life Technologies) supplemented with 66 mmol/L biotin, 34 mmol/L D-pantothenate (Sigma-Aldrich), 10% fetal calf serum (Bodinco BV) and 1% penicillin and streptomycin (Life Technologies) as described before [27 ]. At passage 9 cells were seeded in 6-well plates (Corning Life Sciences, Amsterdam, The Netherlands) with the amount of 3 × 104 cells per well. Every two days, the medium was refreshed. Once the pre-adipocytes reached 90% confluence (T0), the medium was changed to serum-free DMEM/F12 differentiation medium containing 2 mg/mL human transferrin, 200 µmol/L human insulin, 5 mmol/L cortisol, 20 µmol/L triiodothyronine, 1 mmol/L 3-isobutyl-1-methylxanthine and 5 mmol/L rosigilitazone (Sigma-Aldrich). After 4 d the medium was changed to serum-free DMEM/F12 medium containing 2 mg/mL human transferrin, 200 µmol/L human insulin, 5 mmol/L Cortisol, 20 µmol/L triiodothyronine. Every second day, the differentiation medium was refreshed. After 14 d, 85–88% of pre-adipocytes were differentiated into mature adipocytes (T14). To determine cell numbers, pre-adipocytes were trypsinized and counted with a haemocytometer (Countess), adipocytes were counted using a raster ocular.
7
Isolation of Adipose Stromal Vascular Fraction
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Inguinal white adipose tissue was surgically removed from mice, then minced and digested with collagenase II in 0.5% BSA in PBS at 37°C with agitation. The digestion was quenched by adding AT buffer (0.5% BSA in PBS). Dissociated cells were filtered through a 100 μmol/L filter and centrifuged at 500 × g for 10 minutes. The supernatant containing mature adipocytes was aspirated, and the pellet, consisting of the stromal vascular fraction, was resuspended in red blood cell lysis buffer for 2 minutes at room temperature. The reaction was stopped by adding AT buffer and centrifugation at 500 × g for 10 minutes. Cells were washed in 2 mL magnetic-activated cell sorting (MACS) buffer (0.5% BSA and 2 mmol/L EDTA in PBS) and labeled with biotin-conjugated antibodies against lineage markers of endothelial cells (anti-CD31; clone MEC13.3; #102503), immune cells (anti-CD45; clone 30-F11; #103103), and erythrocytes (anti-TER119; clone TER-119; #116203). Cells were then incubated with Streptavidin MicroBeads (Miltenyi, #130–048–101) and subjected to MACS. The lineage depleted cells were harvested and maintained in DMEM/F12, supplemented with 1% GlutaMAX-I, 1% penicillin–streptomycin (all Gibco/Life Technologies), 10% FCS (Biochrom), 33 mmol/L biotin (Sigma), and 17 mmol/L D-pantothenate (Sigma) at 37°C with 5% CO2. All antibodies were purchased from BioLegend.
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