10 mM ATP Stock
(from Maniotis)

• 60 mg ATP in 8 mL H2O
  
• pH to 7.0 with 0.1 M NaOH
  
• Bring volume to 10 mL with H2O
  
• Aliquot and freeze.

Sigma A9525 1mg/mL in water

Vasoconstrictor

Soluble in water 25 mg/mL (only use sterile water!)

MW: 1046.18 Asp-Arg-Val-Tyr-Ile-His-Pro-Phe

Subject to degradation in freezer storage - wear gloves!

Make 1 mg/mL stock (= 1.0462 mM)

Working concentration 1 µM

Dissolve 1:1000 in HBS

BMS 833923

Cayman Chemical 16240

Smoothened (Smo) is a cell surface receptor that, with Patched, mediates sonic hedgehog (Shh) signaling to regulate gene expression through the Gli transcription factors.1 BMS 833923 is an orally bioavailable inhibitor of Smo.2,3 It blocks binding of BODIPY cyclopamine (IC50 = 21 nM) and inhibits Gli activation in cell lines that express wild-type Smo or activated mutant forms of Smo (IC50s = 6-35 nM). BMS 833923 robustly inhibits Shh pathway activity and prevents tumor growth in medulloblastoma and pancreatic carcinoma xenograft models.

References

1. Ruiz-Gómez, A., Molnar, C., Holguín, H., et al. The cell biology of Smo signalling and its relationships with GPCRs. Biochim. Biophys. Acta1768(4), 901-912 (2007).

2. Sandhiya, S., Melvin, G., Kumar, S.S., et al. The dawn of hedgehog inhibitors: Vismodegib. J. Pharmacol. Pharmacother.4(1), 4-7 (2013).

3. Lin, T.L. and Matsui, W. Hedgehog pathway as a drug target: Smoothened inhibitors in development. OncoTargets and Therapy5, 47-58 (2012)

Bisphenol A

Sigma 239658

MW 228.29

100 mM stock in DMSO = 0.023g/mL

Sulfonyldiphenol

Sigma 103039

MW = 250.27

100 mM stock in DMSO = 0.025g/mL

Myosin inhibitor

(-)-Blebbistatin Sigma B05650-1mg

Info from Cayman:

A stock solution may be made by dissolving the (±)-blebbistatin in an organic solvent purged with an inert gas. (±)-Blebbistatin is soluble in organic solvents such as DMSO and dimethyl formamide (DMF). The solubility of (±)-blebbistatin in these solvents is approximately 10 mg/mL.

(±)-Blebbistatin is sparingly soluble in aqueous buffers. For maximum solubility in aqueous buffers, (±)-blebbistatin should first be dissolved in DMF and then diluted with the aqueous buffer of choice. (±)-Blebbistatin has a solubility of approximately 0.5 mg/ml in a 1:1 solution of DMF:PBS (pH 7.2) using this method. We do not recommend storing the aqueous solution for more than one day.

(±)-Blebbistatin is a selective cell-permeable inhibitor of non-muscle myosin II ATPases.1,2 It rapidly and reversibly inhibits Mg-ATPase activity and in vitro motility of non-muscle myosin IIA and IIB for several species (IC50 = 0.5-5.0 μM), while poorly inhibiting smooth muscle myosin (IC50 = 80 μM).3 Through these effects, blebbistatin blocks apoptosis-related bleb formation, directed cell migration and cytokinesis in vertebrate cells. Blebbistatin is inactivated by UV light,4 which may be particularly important in fluorescent cell imaging applications.

Note green fluorescent crystals seen in a 75µM solution.

Stock was 1 mg/mL in DMSO

44 µL 1mg/mL blebbistatin in DMSO in 1 mL non-CO2 medium

REDO

1 mg in 34 µL DMSO = ~100mM

MW = 292

Use at ~150 µM

• 9-amino acid peptide chain: Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg

Bradykinin raises internal calcium levels

*Sigma B3259 - 1 mg septum bottle

• MW = 1060.2
  

  1 mg/mL stock
  (= 1.0602 mM)

Add 1 mL H2O to the 1 mg in the bottle (NO CALCIUM! - some will be used for the calcium experiments in Lab 7.2)

Aliquots are 21.2 µL.

2 µM solution for use
(=2.12 µg/mL)

Fisher 00-4506 1000x solution

Keep in refrigerator

Description

Brefeldin A is an inhibitor of intracellular protein transport. Incubation of cells in culture with Brefeldin A leads to blockade of protein transport to the Golgi complex (GC) and accumulation of proteins in the endoplasmic reticulum (ER). Addition of Brefeldin A during the last hours of in vitro activation of cells results in enhanced detection of intracellular cytokines. Brefeldin A is effective for enhanced detection of a majority of mouse and human intracellular cytokines; however, it is advised that the investigators evaluate the use and efficacy of this reagent as well as other protein transport inhibitors such as Monensin in their specific assay system.

Applications Reported

The Brefeldin A Solution is supplied at 1000X working concentration in methanol. Prior to addition to the cell cultures, a 1:1000 dilution in the culture media should be made. The final working concentration of the Brefeldin A (at 1x) is 3.0 ug/ml.

MW 194.19

Soluble 10 mg/mL in water (with heat and stirring)

Make a 50mM (=0.05M) solution: 9.71mg/mL

Tetrahydrocannabinol solution 1 mg/mL in methanol

Sigma T464

store at 4C

Vial is in a white box in fridge door, room 362A

PLK4 (Polo-like kinase) inhibitor

Plk4 has been identified as a master regulator of centriole replication, and its aberrant expression is closely associated with cancer development

PLK4 functions downstream of ROCK2 to drive centrosome amplification in arrested cells. PLK4 overexpression induces centrosome amplification and chromosome instability and causes the suppression of primary cilia formation.

100uM stock

• use 125 nM for HeLa (2.5 uL stock in 2 mL medium)

• use 300 nM for LLCPk-1 (6 uL stock in 2 mL medium)

• Sigma C5275 5 mg
• Plant mitogen
• Lectin (carbohydrate-binding protein) extracted from the jack-bean, Canavalia ensiformis

• Con A binds specifically to α-Mannose, α-Galactose structures found in sugars, glycoproteins and glycolipids

• Make stock 5 mg/mL solution (add 1 mL sterile PBS to bottle – gently rotate to mix)

• Keep sterile - work in sterile hood

• aliquot 100 µL into microfuge tubes  (cannot take repeated freeze-thaw cycles)

CKD1 inhibitor IV RO3306

1 mM in DMSO

A cyclin-dependent kinase inhibitor protein inhibits cyclin-dependent kinase. Several function as tumor suppressor proteins. Cell cycle progression is negatively controlled by cyclin-dependent kinases inhibitors (called CDIs, CKIs or CDKIs). CDIs are involved in cell cycle arrest at the G1 phase.

Protein synthesis inhibitor

Fisher AC357420010

MW = 281.35 g/mol

C15H23NO4

Solubility in water: 2.1 g/100mL (

soluble in chloroform, ether, acetone, methanol, and ethanol

powder

ApexBio A8340

10 mM

Store in freezer

C27H41NO2

MW = 411.62

Soluble 6.86 mg/mL in DMSO (warm to 37C and shake to get it into solution)

Reaction Conditions :

• 20 μM, 48 hours for cell yield inhibition
• 10 μM, 48 hours for apoptosis induction (measured by PARP expression)

Cyclopamine is a naturally occurring Hedgehog (Hh)?specific small?molecule signaling steroidal alkaloid inhibitor, causes a profound inhibition of tumor growth, has significant anti?invasive, anti?proliferative and anti?estrogenic potency in human breast cancer cells [2] [1]. The EC50 of cyclopamine is 10.57 μM, it was identified by an FXR-bla (farnesoid X receptor- b-lactamase) assay [3].

Hh signaling pathway plays a critical role in embryonic development and tumorigenesis [4]. Hh signaling pathway shows saliency in regulating cellular proliferation and differentiation in a wide array of human tissues. It is related to aberrant cell survival in numerous human malignancies, ranging from BCCs and medulloblastomas to small cell lung, gastrointestinal, breast and prostate tumors [1].

Treated with cyclopamine (10 or 20 μM) only and incubated for time periods ranging from 0 to10 days, MCF-7 cells and MDA?MB?231 cells displayed a significant reduction in proliferation rate compared with the control cells on days 3 and 6 (P

Embryoes exposed to cyclopamine resulted in visible external defects, including cyclopia, proboscis formation, microphthalmia, thoracic lordosis, amelia and decreased body size. Examination of gastrointestinal organs revealed severe deficits, including less length of the gut tube and mesenchymal cell numbers in foregut-derived organs. Ectopic structures in duodenum, stomach, and dorsal pancreas were also found [5].

MW = 507.62
Sigma C2618: 200 µL 5 mg/mL (~10 mM) in DMSO
Sigma C8273: 5 mg. Dissolve in 1 mL DMSO

• alkaloid mycotoxin produced by Helminthosporium and other molds.
• disrupts actin microfilaments
• binds to F-actin polymer
• prevents polymerization of actin monomers
• Aliquot 10 µL 10 mM
• Add 90 uL DMSO (makes 1 mM)
• Dilute this 1:1000 into medium (makes 1uM)

• Dilute the 1 uM to desired working concentration

• Store in the dark, in the freezer

• Adding 490 µL of non-CO2 medium makes 500 µL of 200 µM

• Final dilution should contain no more than 0.1% DMSO

• 250 nM in the dish should give an effect

• 15 - 30 min

Biological Activity for DAPT (Info from Tocris 2634)

Purchased from Fisher: AAJ65864MA

Mfr: Thermo Scientific Chemicals J65864MA

M. Wt: 432.46 Formula: C23H26F2N2O4 Store at +4°C Light sensitive

Solubility: 43.24 mg/mL (100 mM) in DMSO

DAPT is a γ-secretase inhibitor. DAPT reduces Aβ40 and Aβ42 levels in human primary neuronal cultures (IC50 values are 115 and 200 nM for total Aβ and Aβ42 respectively) and in brain extract, cerebrospinal fluid and plasma in vivo. DAPT has no effect on APPα and APPβ levels. DAPT blocks Notch signaling in hybrid human-mouse fetal thymus organ culture (FTOC) and causes ESCs to commit to neuronal differentiation. DAPT can be used in a small molecule cocktail to derive cortical neurons from hPSCs and to maintain hepatocytes in culture. DAPT also promotes the formation of cone photoreceptors in retinal organoids.

β17-Estradiol

Cayman Chemical 10006315

(Purchased 2022 for NAP lab Bio 388)

500 mg

Store powder at -20

Make 0.05M stock solution: 0.068 g in 5 mL DMSO. vortex

Estrogens direct the development of the female genotype in embryogenesis and at puberty. 17β-Estradiol is the major estrogen secreted by the premenopausal ovary. It is synthesized from testosterone primarily in the ovarian granulosa cells and placenta, but small amounts can be produced in the adrenal gland.1,2 Plasma 17β-estradiol levels increase gradually between days 1-7 of the menstrual cycle followed by a sharp increase to a peak value of about 300 pg/ml on day 12, just prior to ovulation

Sigma F1141

Attachment factor

Smo (Smoothened) receptor* agonist

Tocris 4918 10 mg

MW = 459.94

Make a 5 mM stock solution in 4.34 mL DMSO (heat to 50C to dissolve)

*Non-classical G-protein-coupled receptors that belong to the Frizzled family. Smoothened receptors lack the ability to directly interact with their endogenous ligand, Hedgehog (Hh). In the resting state, Smo receptors are bound to patched (Ptc).

Does not recognize the classic cyclopamine (Cat. No. 1623) binding site. Does not promote Smo translocation to the primary cilium; is strongly potentiated by forskolin and cholera toxin. Displays anti-adipogenic effects in vitro, mediated by a non-canonical Hedgehog signaling pathway. Promotes differentiation of multipotent mesenchymal progenitor cells into osteoblasts.

H 89 hydrochloride Tocris 2910

• Protein kinase A inhibitor
• Also inhibits several other kinases (IC50 values are 80, 120, 135, 270, 2600 and 2800 nM for S6K1, MSK1, PKA, ROCKII, PKBα and MAPKAP-K1b).
• Exhibits antinociceptive activity.
• Enhances survival and clonogenicity of dissociated human ESCs through ROCK inhibition.

Store in freezer

1 mg

MW 532.79

1 mg/mL in DMSO

data sheet

Hydroxyurea: (Sigma H8267-1g)

• To make 50 mL YPD with 0.2M HU
  

• vi = (0.2M * 50 mL)/2M = 5 mL

• 100µL 2M stock per mL medium

• Buy 1 g, make 6.6 mL 2M stock
  

• MW = 76, so 1 g = 1/76 mol = 0.0132 mol
  
• vol = (1000 mL/2 mol) * 0.0132 mol = 6.6 mL

See yeast recipes for QBoC https://wahoo.cns.umass.edu/node/554/

TCI H0912

C21H23CIFNO2

MW 375.87

Solubility:¹

• 0.1 M HCl: 3 mg/mL
• DMSO: soluble
• H2O: insoluble
• ethanol: soluble (only in theory - in practice, not so much)

Make 10 mL 10mM stock solution: 0.376g in 10 mL DMSO. Vortex to mix

Haloperidol has been used:

• in ethanol to serves as an inhibitor of Erg2p
• to address the mechanism of haloperidol in ferroptosis using hepatocellular carcinoma cells: Hep G2 and Huh-7 cell lines
• in receptor internalization assay
• as an antipsychotic drug in Dulbecco′s Modified Eagle medium

Biochem/physiol Actions:

Haloperidol is a butyrophenone antipsychotic. It is also classified as a neuroleptic (powerful tranquilizer). Haloperidol acts as a D2, D3, and D4 dopamine receptor antagonist and thus causes Parkinson′s disorder. It also has a negative effect on the central nervous system.

Fisher A16292 (Alfa Aesar) 1 g

1 mg/mL in 100% ethanol.

Insulin-like growth factor-1 receptor inhibitor

Selleck Chemicals BMS-536924

Molecular Formula: C25H26ClN5O3

Formula Weight: 479.96

Solublity: 96 mg/mL in DMSO

Selleck Chemical S7085 (Fisher NC0736836)

IWP-2 is an inhibitor of Wnt processing and secretion with IC50 of 27 nM in a cell-free assay, selective blockage of Porcn-mediated Wnt palmitoylation, does not affect Wnt/β-catenin in general and displays no effect against Wnt-stimulated cellular responses. IWP-2 specifically inhibits CK1δ.

Selective inhibitor of Porcn-mediated Wnt secretion.

IWP-2 is useful in both regenerative medicine and anticancer efforts. IWP-2 inactivates Porcn, a membrane-bound O-acyltransferase (MBOAT), and selectively inhibits palmitoylation of Wnt. IWP-2 blocks Wnt-dependent phosphorylation of Lrp6 receptor and Dvl2, and β-catenin accumulation.

Soluble in DMF at 12.5 mg/mL (26.79 mM) Insoluble in water and DMSO

401105-10mg Calbiochem

Importazole is an inhibitor of importin-β transport receptors.

During interphase, the transport receptor importin-β carries cargoes into the nucleus, where RanGTP releases them. Importazole somehow disrupts the importin/RAN interaction. Importazole is selective among other transporters. Compounds are imported into, but not out of, cells.

A cell-permeable diaminoquinazoline compound that selectively blocks importin-β-mediated nuclear import of NLS bearing cargos in a reversible manner.

Soluble in DMSO (25 mg/ml; clear, colorless solution)

5 mg/mL ailquots in freezer

Raises intracellular calcium levels

Fisher or Invitrogen (Life Technologies) I24222

1 mM in DMSO or EtOH. 1 mg makes 1.34 mL stock.

3 µM Ionomycin in HBS
Dilute stock 1:333 in HBS: 3 µL/1 mL

24 µL in 8 mL, aliquot 800 µL

They only need 750µL, because they do one experiment in HBS, then two in Ca-free HBS.

3 µM Ionomycin in Ca-free HBS
Dilute stock 1:333 in Ca-free HBS: 3 µL/1 mL
45 µL in 15 mL

Takes a long time to thaw. Vortex.

Aliquot 1.55 mL so they can do two 750 µL experiments

(out of stock 2018)

• Jasplakinolide – Santa Cruz biochemical, product sc202191 F
  • inducer of actin polymerization & stabilization; inhibits actin filament disassembly
  • F-actin probe
  • MW = ~710
  • powder is stable frozen over a year
  • stock is stable 3-4 mo at -20C
  • Make 1 mM stock with 50 µg in 70 µL DMSO
  • Add 5 mL non-CO2 medium to make ~14 µM
  • 50 nM to 5 μM working range
  • minutes to hours incubation time

Low-density lipoprotein from human plasma, Dil complex (Dil LDL)

1 mg/mL

200 uL

DO NOT FREEZE

Life Technologies L3482

Laminin from mouse BD354232

1 mL 1 mM

Cell adhesion

Alexis Biochemicals 350-036-C100 Catalog # t110

Inhibits actin polymerization and disrupts microfilament organization, as well as microfilament-mediated processes

Reported to be 10 to 100-fold more potent than the cytochalasins. May act more slowly, though. Lots of variation between cell lines.

Inactivated by FBS. (Rinse medium off before treatment.)

MW = 395.5

Soluble in DMSO and ethanol.

Store in freezer.

Active concentration range: 90 nm ( =~0.1µM) to 2.5 µM

(2.5 µM = 25 x 100 nm)

Stock is 1mm

*Use this to make further dilutions

ML-7 Sigma, 12764.

selective myosin light chain kinase inhibitor

• soluble 10mg/mL in 50% EtOH
• MW = 452.74
• 5 mg in bottle
• add 1100 µL to make 10 mM solution
• used at ~50 µM (1 µL/1mL) (Pat says 75 µM)
• 75 µL 10 mM + 5 mL non-CO2 medium makes 150 µM
• make 15 µL aliquots of 10 mM. Add 985 µL medium to make 150 µM

Sigma M4135 1 mg

Molecular Weight 1664.88

From Sigma: Amino Acid Sequence:

Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2

Pituitary hormone which causes darkening skin pigmentation from amphibians to humans. In mammals, it can also have behavioral effects on learning, attention, and memory.

Hormone that stimulates melanogenesis; facilitates learning and memory; affects inflammatory and immune responses and peripheral nerve regeneration.

α-Melanocyte-stimulating hormone (α-MSH) acts as an anti-inflammatory agent via down regulating the production and activity of the pro-inflammatory cytokines interleukin-1 (IL-1), tumor necrosis factor (TNF)-α and IL-6 expressed in various cells of the immune system. It also controls the nitric oxide production associated with inflammation. α−MSH inhibits nuclear factor-κB (NF-κB)-dependent gene transcription and NF-κB pathway induced by TNF and other inflammatory agents. This activity of α-MSH is mediated through the production of cyclic adenosine monophosphate (cAMP) and activation of protein kinase A (PKA) enzyme. α–MSH functions as a potent therapeutics for various conditions resulted through NF-κB activation including, inflammatory diseases, human immunodeficiency virus (HIV) replication in AIDS (acquired immunodeficiency syndrome), and septic shock.[2] α-MSH has an essential role to play in melanin production in animals. α-MSH regulates development of several skin diseases, including cutaneous inflammation and hyper-proliferative skin diseases.[3] Linkage

Derived from ACTH 1-13 Other Notes

Lyophilized from 0.1% TFA in H2O Packaging

1, 5 mg in glass bottle Application

α-Melanocyte stimulating hormone (α–MSH) has been used for following studies: • it has been Intracerebroventricularly (icv) injected to mice for behavioral studies.[1] • to determine the effect of α−MSH on growth of stationary Nb 2 node lymphoma cell cultures.[4] • to determine the effect of α−MSH on leptin secretion in the primary cultures of differentiated adipocytes.[5] • α−MSH promoted melanin production in the B16-F1 cells from murine melanoma cell line.[6] General description

α-Melanocyte-stimulating hormone (α-MSH) is a tridecapeptide, mostly produced by the cells in the brain, pituitary and circulation.[2] Pro-inflammatory cytokines or UV light induced epidermal cells such as keratinocytes and melanocytes synthesize and discharge α–MSH. Poopiomelanocortin (POMC) acts as a precursor for α-Melanocyte-stimulating hormone (α-MSH) production.[3]

Inhibits kinesin 5

200 mM in DMSO

Final concentration 200 uM

5.5 uL aliquots

Monensin sodium salt

Sigma M5273

soluble in methanol 50 mg/mL

insoluble in water

soluble in DMSO, ethanol

ionophore which disrupts the structure of the Golgi apparatus and inhibits vesicular transport in eukaryotic cells

inhibits transition from G1 to S

antiprotozoal, antibacterial, or antifungal agent

MW 692.9

1 µM = half-maximal inhibitory effect

Nerve Growth Factor 2.5S

Harlan.com Cat no. 5025

From Sigma: 2.5S subunit of nerve growth factor-7S (NGF-7S) is essentially the β-subunit when isolated from male mouse submaxillary glands under initially dissociative conditions by a modification of the method of Bocchini and Angeletti.

Stored at 4C

Prevents polymerization of microtubules. Prevents cells from entering metaphase.

• Acros 358240100, 10 mg
  

• Soluble in DMSO

• Dilute stock in medium to treat cells.

• Make a 10 mg/mL (=33mM) stock: Add 1 mL DMSO to 10 mg in bottle.

• Aliquot 3 µL 33 mM per tube.

• Working concentration range is usually 100nM - 30 µM.

• add 97 µL DMSO to tube to make 100 µL of 1 mM

• 33 µL 1mM + 10 mL medium = 3.3 µM

Oxidopamine (hydrobromide)

Selleck Chemicals S5324-25mg (Fisher 17159569)

MW = 250.09

Formula: C8H11NO3.HBr

Oxidopamine (6-hydroxydopamine, 6-OHDA, 2,4,5-trihydroxyphenethylamine) is a neurotoxic synthetic organic compound that acts as an antagonist of the neurotransmitter dopamine with potential antineoplastic activity. Solutions should be freshly prepared and protected from exposure to light.

PhenylMethylSulfonyl Fluoride

Serine protease inhibitor

Sigma https://www.sigmaaldrich.com/catalog/product/sigma/p7626

Non-specific attachment factor for cells; use it to coat coverslips.

Sigma P1524

1. Add 50 ml of sterile tissue culture grade water to 5 mg of poly-lysine.
2. Aseptically coat culture surface with 1 ml per 25 cm2 of solution. Rock gently to ensure even coating of the culture surface.
3. After 5 minutes, remove solution by aspiration and thoroughly rinse surface with sterile tissue culture grade water.
4. Allow to dry at least two hours before introducing cells and medium. If glassware or slides must be sterilized after coating with poly-lysine, γ-irradiation is recommended instead of autoclaving.

Can also buy poly-lysine coated slides: Fisher 6776215

Tocris 4551 10 mg

MW = 525.12

Make a 10 mM stock in DMSO in 1.9 mL DMSO

Fisher J62079

Store in freezer; hygroscopic

C20H30O

MW = 286.459

General description (from Fisher)

• All-trans-retinol, commonly known as Vitamin A, is a fat-soluble essential nutrient
• All-trans-retinol is involved in hormonal signaling via the retinoid receptors
• It is an important regulator of cell division and apoptosis
• Deficiency can lead to vision, bone, immune, and skin disorders

Practically insoluble aq. soln. or glycerol; soluble in absolute alcohol, methanol, chloroform,ether,fats and oils.

Can make a 50 mg/mL solution in 95% ethanol, but it's very hard to weigh

From Sigma information sheet:

SOLUBILITY / SOLUTION STABILITY:

• RE is practically insoluble in water or glycerol. It is soluble in absolute ethanol, methanol, chloroform, ether, fats and oils.
• RE has been dissolved at 50 mg/ml in chloroform; a clear yellow to orange solution results.
• Stock solutions of RE (1 mg/ml) were prepared in ethanol, diluted in DMSO under low light conditions and stored at -50°C under nitrogen in brown glass vials.
• RE solutions (50 μM) were sterile filtered before use.
• RE both as a solid and in solution is readily oxidized in air and inactivated by UV light.
• To reduce photodestruction of RE, manipulations of RE solutions can be performed under yellow or red light.
• Solutions may be stabilized by dissolving in oil, by the addition of anti-oxidant compounds including a-tocopherol or hydroquinone or by conversion to the palmitate and acetate esters.
• It is recommended to prepare solutions fresh for optimal quality. However, if absolutely necessary, store solutions in the dark under an inert atmosphere at least at -20°C preferably at -70°C
• Solvents preferred for storage are peroxide-free ethyl ether, acid-free acetone or ethyl acetate.
• For short term storage, ethanol is suitable as a solvent for spectroscopic analysis.

USAGE / APPLICATION

• The isolation of retinol from human plasma has been described.
• RE is an effective antioxidant displaying lipoperoxy radical scavenging activity.
• The interactions between RE and Vitamin E (a-tocopherol) in suppressing lipid peroxidation were observed in bovine retinal membrane preparations.
• RE may influence the production of transition vesicles by stimulating the activity of a protein disulfide isomerase-like activity involved in vesicle formation.
• RE may be involved in immune system mechanisms; an RE deficiency will depress the immune response producing a negative effect on both humoral and cellular immunity.
• (10 μM) and other retinoid compounds effectively induced sanguinarine and chelerythrine (benzophenanthridine alkaloids) accumulation in suspension-cell cultures of Sanguinaria canadensis in a way similar to fungal elicitation.
• RE (10 μM) stimulated DNA synthesis and possibly repair mechanisms in Sertoli cells of rat.

GENERAL NOTES

The USP unit of vitamin A (same as the International Unit6) is equal to 0.3 μg of the pure all-trans isomer of retinol which is equivalent to 0.344 μg of all-trans retinyl acetate. RE and its metabolites, including retinoic acid, are part of the retinoid class of compounds, involved in vision, normal embryo morphogenesis and in the regulation of proliferation and differentiation of a number of cell types. Current information and hypotheses on the absorption, transport, storage and metabolism of this fat soluble Vitamin A (retinol) have been reviewed. Studies on RE metabolism including its mobilization and transport in plasma and in tissues via serum and cytosolic retinol-binding proteins have been described.

• STLC Sigma 164739 (Fisher 50-703-1833), MW = 363.47

(+)-S-Trityl-L-cysteine is a cell-permeable selective inhibitor of mitotic kinesin Eg5 and ATPase activities.

KIF11 (also known as kinesin-5 and Eg5) is a homotetramer which cross-links anti-parallel microtubules in the mitotic spindle to maintain spindle bipolarity. The motor domain or motor head is at the N-terminus and performs ATP hydrolysis and binds to microtubules. (https://en.wikipedia.org/wiki/Kinesin_family_member_11)

• Make 100 mM stock:

  • 1 g in bottle.
  • Mix with 27.5 mL DMSO
  • heat to 65C (~10 min), vortex
  • if necessary, filter sterilize to remove insoluble particles.
  • save as 1 mL aliquots in freezer. (dilute 1:10,000 to use) (dilute 1:100 to make 1 mM)

• Make 1 mM stock from the 100 mM stock

  • 10 µL 1mM stock
  • 990 µL DMSO
  • vortex
  • it will crystallize in the refrigerator. Warm (60C) and vortex to redissolve it. DO NOT LEAVE IN HOT BLOCK - IT WILL TURN BLACK!

• Make 10 µL aliquots of 1 mM stock. Freeze

  • label says to add 990 µL medium to make 10 µM working solution

ALGINIC ACID SODIUM SALT 5G

Fisher 177770050 $22.41

(Can get 100g from Sigma for $55, but with long lead time)

(C6H7O7)A(C6H7O7)BNa

MW=120,000-190,000 g/moL

The following is from Sigma:
General description

Alginic acid sodium is a gelling and nontoxic anionic polysaccharide. The carboxylic acid groups on the alginic acid chain, renders it insoluble in water.However, converting alginic acid to its sodium form, enables it to solubilize in water easily.

Application

Alginic acid sodium is used:

• in combination with chitosan, to fabricate a biodegradable porous scaffold for bone tissue engineering.
• to study the characteristics of a modified amphiphilic alginate derivative
• to the study the impact of alginate on the rate of lipid digestion by employing an in vitro digestion model
• in the preparation of alginate hydrogels
• as encapsulating agents of microparticles of β-galactosidae

Restek 34067

Delta-9 THC Standard

1 mg/mL in methanol (=~3.18 mM)

C21H30O2

MW = 314.469

Taxol (20 µM final)

Fisher 109710 Paclitaxel.
Sigma T7402 1mg
MW = 853.91

• Stabilizes the microtubule polymer
• Protects microtulues from disassembly.
• Prevents metaphase spindle configuration

• Blocks progression of mitosis and prolonged activation of the mitotic checkpoint triggers apoptosis or reversion to the G-phase of the cell cycle without cell division.

• soluble in DMSO, not water.

• Add 0.117 mL DMSO to make a 0.01M (=10 mM) stock solution from the 1 mg powder

• Aliquot 10 µL 10 mM (if it precipitates, add 10 uL DMSO and vortex, then add 480 uL medium or buffer)

  • add 490 µL to make 200µM
  • dilute into medium from there (100 uL Taxol/mL medium)

for Fish

5 mL per well for treatment

PTU (inhibitor) Sigma p3755
MW 170.2
Solubility:

• 50 mg/mL in 1M NaOH
• 16 mg/mL in alcohol
• 1 mg/mL in RT water
• 10 mg/mL in boiling water

In order to get the following doses, best to make it in hot fish water; otherwise, the NaOH or EtOH will be too concentrated in the final dilution.

• dose 1: 0.5 mM

• dose 2: 1.0mM

L-T4 (thyroxin) Sigma T2376
MW: 776.87
solubility:

• 50 mg/ml in 4M NH4OH in MeOH
• 0.5 mg/mL in 1:5 DMSO:PBS

Solution is good for ~ 1 day.

Make a 1 mM stock in 1M NaOH. Takes a long time. Vortex.

Priya's recipe: 1 mg/mL in 1M NaOH (~25.7 uM stock)

Working concentrations

• dose 1: 100 nM (=0.1 uM)

• dose 2: 300 nM (=0.3 uM)

Cayman 89730

10 mM

Histone deacetylase Inhibitor

Trichostatin A (TSA), an antifungal antibiotic produced by Streptomyces hygroscopicus, is a potent and specific inhibitor of histone deacetylases (HDACs), which are overexpressed in various cancers and closely correlate with oncogenic factors.

Trichostatin A is active at nanomolar concentrations in mammalian cells. By suppressing the activity of HDACs, it leads to increased histone acetylation, thereby causing highly acetylated histones to accumulate in the cell [2]. This in turn induces enhanced expression of specific genes that elicit extensive cellular morphologic and metabolic changes such as growth arrest, differentiation and apoptosis. At submicromolar concentrations Trichostatin A has been shown to induce apoptosis in diverse cancer cells while exhibiting very low toxicity to normal cells.

TSA is sparingly soluble in aqueous buffers. For maximum solubility in aqueous buffers, TSA should be directly dissolved in 0.1 M HCl (1.3 mg/ml) and then neutralized with PBS (pH 7.2) to achieve the desired concentration or pH. Approximately 0.7 ml of PBS (pH 7.2) is required to neutralize 1 ml of the acidic solution. We do not recommend storing the aqueous solution for more than one day.

Potassium Ionophore, MW: 1111.32

Sigma V0627

Insoluble in water

stock is 10 mg/mL in DMSO

• cyclododecadepsi-peptide ionophore antibiotic.
• potassium ionophore that transports K+ across biological and artificial lipid membranes.
• can induce K conductivity in cell membranes
• uncouples oxidative phosphorylation,
• induces apoptosis in murine thymocytes, and in pre-B cell.
• inhibits NGF-induced neuronal differentiation
• antagonizes ET-induced vasoconstriction.
• Useful in studies of K transport in mitochondria.

Sodium Valproate (Fisher 11474361)

Store powder at RT

Make 100 mM stock: 0.166g in 10 mL H2O

(=Anti-diuretic hormone)

causes calcium oscillations

1 mM stock in water
(NO CALCIUM, so stock can be used for Ca-free solutions in Lab 7.2)

1 mg in bottle, MW = 1084
Add 0.92 mL to make 1mM stock

1 µL aliquots in freezer

1 µM dilution to make student solutions

1 µL 1 mM in 999 µL water

(NO CALCIUM, so stock can be used for Ca-free solutions in Lab 7.2)

Thermo Scientific B22524.03

D3-calciferol

C27H44O

MW = 384.65

Solubility (from Cayman Chemical)

Stock solution: 29 mg/mL (~75 mM) in ethanol

General Description (from Thermo Scientific)

• Vitamin D3 is a steroid hormone generated in the skin when the 7-dehydrocholesterol interacts with ultraviolet irradiation. It can also be found in several types of food for daily intake.
• Vitamin D3 binds to vitamin D receptors, thus modulating gene expression

Applications

• In in vivo studies, vitamin D3 has been implicated in the maintenance of blood calcium and phosphorus levels, bone metabolism, metabolic functions, and transcription regulation
• It modulates the proliferation and differentiation of both normal and cancer cells
• It presents in vitro antiproliferative and antimetastatic activities on breast, colon, and prostate cancer cells