Mice were injected subcutaneously with 2 mg Medroxyprogesterone 17-acetate (Sigma) 4 days prior to intravaginal inoculation with 20 μg LPS from Salmonella enterica serotype Minnesota (Sigma), HSV-2 alone, 108 recombinant C. crescentus alone, or HSV-2 plus C. crescentus. Vaginal inoculation was performed in a volume of 20 μL using a sterile p200 pipette. Vaginal lavage was performed 4 days prior to and 4, 24 and 48 hours post-inoculation by pipetting twice consecutively with 30 μL PBS. Vaginal lavage fluid was centrifuged at 400 × g and the supernatant was stored at −80 °C until analysis. 25 μL vaginal lavage fluid was analyzed using a Cytometric Bead Array kit for Mouse Inflammation (BD Biosciences, Catalog No. 552364) that detects mouse IFNγ, TNF, IL-6, IL-10, MCP-1, IL-12p70 according to the manufacturer’s instructions. Data was acquired on an LSRII and analyzed using FlowJo (TreeStar) and Prism. Briefly, mean fluorescence intensity for each cytokine was determined in FlowJo and experimental values were interpolated from the standard curve created using the BD CBA Mouse Inflammation Standards.
Medroxyprogesterone 17 acetate
Manufactured by Merck Group
Sourced in United States
Medroxyprogesterone 17-acetate is a synthetic progestin compound used in various laboratory applications. It functions as a progesterone receptor agonist, exhibiting progestogenic activity. This compound is commonly utilized in research and development settings.
Automatically generated - may contain errors
Lab products found in correlation
Lps from salmonella enterica serotype minnesota, by Merck Group (1 mentions)
Total rna isolation kit, by Thermo Fisher Scientific (1 mentions)
8 bromoadenosine 3 5 cyclic monophosphate sodium salt, by Merck Group (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Dmem f12, by Merck Group (1 mentions)
Ab141058, by Abcam (1 mentions)
Crl 4003, by American Type Culture Collection (1 mentions)
Dbcamp, by Merck Group (1 mentions)
Dmem f12, by Sartorius (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Ethanol, by Merck Group (1 mentions)
Chloroform, by Merck Group (1 mentions)
Nuclease free water, by Thermo Fisher Scientific (1 mentions)
Tris edta buffer, by Merck Group (1 mentions)
Qunatitect reverse transcription kit, by Qiagen (1 mentions)
8 bromo cyclic adenosine monophosphate camp, by Merck Group (1 mentions)
Estrogen, by Merck Group (1 mentions)
5 protocols using medroxyprogesterone 17 acetate
1
Vaginal Immune Response to Microbial Challenges
2
Decidualization of Human Endometrial Stromal Cells
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HESCs isolated from the proliferative phase of the menstrual cycle were grown in a six-well culture plate at 60–70% confluence and transfected with 60 pmol of nontargeting siRNA (D-001810-10-05) or siRNAs targeting ACE2 (L-005755-00-0005) (GE Healthcare Dharmacon Inc., Lafayette, CO) in Lipofectamine 2000 reagent (Invitrogen Corporation, Carlsbad, CA), as described previously [15 (link)]. After 48 h, HESCs were decidualized by culturing in EPC (Estrogen, Medroxy Progesterone Acetate and cAMP) medium (1x Opti-MEM reduced-serum media containing 2% FBS, 100-nM estradiol [cat. no. E1024, Sigma-Aldrich, Saint Louis, MO, USA, 10-μM Medroxyprogesterone17-acetate [cat. no. M1629, Sigma-Aldrich, Saint Louis, MO, USA], and 50-μM 8-Bromoadenosine 3′,5′-cyclic monophosphate sodium salt [cat. no. B7880, Sigma-Aldrich, Saint Louis, MO, USA]). The EPC medium was changed every 48 h until day 6, when the cells were harvested for RNA isolation with the total RNA isolation kit (Invitrogen/Life Technologies, Grand Island, NY) or for protein isolation.
3
Decidualization of Human Endometrial Stromal Cells
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Immortalized human endometrial stromal cells (hESC) were purchased from the American Type Culture Collection (ATCC, CRL-4003TM) and cultured according to the manufacturer's instructions. Briefly, stromal cells were cultured in DMEM/ F12 (Sigma-Aldrich) supplemented with 10% charcoalstripped FBS (cFBS, Biological Industries, Invitrogen) at 37°C in a humidified chamber with 5% CO 2 . Decidualization in vitro was induced as previously described (Liang et al. 2018) (link). Stromal cells were treated with 1 μM medroxyprogesterone 17-acetate (Sigma-Aldrich) and 0.5 mM dibutyryl cAMP (db-cAMP, Sigma-Aldrich) in DMEM/F12 with 2% (v/v) cFBS (Biological Industries) for 6 days. The medium was changed every 48 h. Under the condition of decidualization and non-decidualization, stromal cells were treated with 3.5 nM ActD (ab141058, Abcam) for further analyzing the effects of nucleolar stress on in vitro decidualization.
4
Quantification of PRL gene expression
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8-Bromo-cyclic adenosine monophosphate (cAMP) and medroxyprogesterone 17-acetate (MPA) were obtained from Sigma Aldrich. Nuclease-free water was obtained from Ambion. QunatiTect Reverse Transcription Kit was obtained from QIAGEN. Chloroform, isopropanol and ethanol were obtained from Sigma Aldrich. Tris-EDTA buffer was obtained from Sigma Aldrich. SyBr Green was obtained from Applied Biosystems. Forward and reverse PCR primers for PRL and L19 were obtained from Peprotech.
5
Modeling Menstrual Cycle Hormones in Cells
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ECC1 cells and pEECs as required, were treated with estrogen in the absence/presence of progesterone to mimic the proliferative and secretory phases of the menstrual cycle respectively [22] . hCG was also added to 'secretory-phase' cultures to mimic the embryonic signal at the time of conception. ECC1 cells (110 6 per 150 mm 3 culture dish, total of 15 dishes/treatment, n = 3 separate experiments) were washed three times with PBS and cultured for 24 hrs in serum-free media (DMEM/F12 supplemented with 0.5% insulin-transferrinselenium (ITS) solution (Invitrogen-GIBCO), and 1% (v/v) Pen/Strep). pEECs (n = 6 separate preparations from individual women, 80% confluence), were similarly washed and cultured for 24 hr in DMEM/F12 supplemented with 0.5% charcoal-stripped (cs) FCS. After 24 hr, media were replenished with the indicated media and all cells primed with estrogen (10 -8 M, Sigma-Aldrich) for 24 hr. Cells were then divided into three groups and treated with (i) E: estradiol-17-beta/estrogen (10 -8 M), (ii) EP: E (10 -8 M) and progesterone (P, medroxyprogesterone-17-acetate, 10 -7 M, Sigma-Aldrich), or (iii) EP and hCG (10 IU/ml) [29, 38] . Conditioned media (CM) were harvested after 24 hr of hormonal treatment and treated as detailed below. Cells were also harvested at the 24 hr hormonal treatment time point.
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