To know the feedback effect of steroids on GnRH, hypothalamuses of female small yellow croaker were incubated with 17β-Estradiol (E2) or 17α-Methyltestosterone (MT) in vitro as described previously [14 (link),15 (link)] with slight modifications. Briefly, E2 and MT (Sigma, USA) were first dissolved in acetone and absolute ethanol, respectively. Hypothalamuses were collected from 30 reproductively mature female fish and washed with M199 medium modified with Hanks’ Balanced Salts (Gibco, Grand Island, NY, USA), transferred to 12-well culture plates, three hypothalamuses in a well and pre-incubated with M199 medium (modified with Hanks’ Balanced Salts) treated with penicillin–streptomycin at 25 °C for 2 h. After pre-incubation, the medium was discarded, and hypothalamuses were washed twice with the same medium. Hypothalamuses were then incubated with M199 medium containing 0.1, 1, and 10 μM E2 or MT for 12 h. A control group was incubated without E2 or MT. After incubation, hypothalamuses were collected at 3 h, 6 h, and 12 h, and stored at −80 °C.
Hanks balanced salts
Manufactured by Thermo Fisher Scientific
Sourced in United States, India
Hanks' Balanced Salts is a commonly used buffer solution designed to maintain the pH and osmotic balance of cell culture media. It contains a combination of inorganic salts, including sodium, potassium, calcium, and magnesium, as well as other components such as glucose and phenol red. Hanks' Balanced Salts is used to maintain the optimal physiological conditions for the growth and maintenance of cells in in vitro cell culture experiments.
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Lab products found in correlation
Medium 199, by Thermo Fisher Scientific (4 mentions)
Trizma hydrochloride solution tris hcl, by Merck Group (3 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Phenylmethanesulphonyl fluoride pmsf, by HiMedia (2 mentions)
N acetyl l leucyl l leucyl l argininal leupeptin, by Avantor (2 mentions)
Cell tak cell and tissue adhesive, by BD (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Statistica for windows, by StatSoft (1 mentions)
I1 hybridoma cells, by American Type Culture Collection (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Leupeptin, by Avantor (1 mentions)
Anti flag m2 affinity beads, by Merck Group (1 mentions)
Anti actin antibody, by Merck Group (1 mentions)
10 protocols using hanks balanced salts
1
Steroid Regulation of GnRH in Female Fish
2
Sperm Motility Analysis in Hgsnat Mice
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Cauda epididymides of 5 Hgsnat+/+ and 8 Hgsnat−/− mice at 11 months were submerged in Medium 199, Hanks’ Balanced Salts (Gibco, cat# 12350–039). Epididymides were cut and squeezed gently to allow spermatozoa to diffuse into suspension for 10 min at 37°C. Sperm concentration was determined with a hemocytometer and then adjusted to 5 × 106 cells/ml in Medium 199, Hanks’ Balanced Salts. Sperm motility parameters were determined using a computer-assisted sperm analysis system (CASA) with Sperm Vision HR software version 1.01 (Minitube, Ingersoll, ON, Canada) [83 (link)]. Two hundred spermatozoa were examined for each sample to determine each sperm motility parameter. Mann-Whitney tests (alternative nonparametric test) were done using Version 8 of Statistica for Windows (Statsoft, Inc., Tulsa, OK). The p values less than 0.05 were considered significant.
3
Cell line maintenance for SARS-CoV-2 research
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Vero-TMPRSS2 cells were generously provided by Dr Benhur Lee (Icahn School of Medicine at Mount Sinai) and are previously described.55 (link) Vero-TMPRSS2 cells were maintained in DMEM (Gibco 11965-092) supplemented with 10% fetal bovine serum (FBS; Gibco 10437-028) and penicillin–streptomycin (1:50; Sigma P4458) at 37°C in a 5% CO2 atmosphere. I1 hybridoma cells (ATCC CRL-2700) were obtained from the ATCC and maintained at 37°C in 100% room air, in minimal essential media with Hank’s balanced salts (Gibco 11575032) supplemented with 2mM L-glutamine (Sigma G7513) and 20% FBS.
4
Pituitary Hormone Regulation by Steroids
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Pituitaries of SYC from both sexes were cultured in vitro with E2 or MT, as described previously [32 (link),47 (link),50 (link)]. Briefly, stock solutions of E2 and MT (Sigma, St. Louis, MO, USA) were prepared using acetone and absolute ethanol, respectively. A working solution for E2 and MT was prepared using 1× PBS. Pituitaries from 80 mature SYC, for each sex, were collected and washed with M199 medium modified with Hanks’ balanced salts (Gibco, Grand Island, NY, USA). Then, the pituitaries were placed in the holes of 12-well culture plates in 3 treatment groups and a control group. Ten pituitaries were placed in each well and preincubated at 25 °C for 2 h in serum-free M199 medium (modified with Hanks’ balanced salts), treated with penicillin–streptomycin. After 2 h of incubation, the M199 medium was disposed of, and the pituitaries were washed twice with the M199 medium. Then, fresh M199 medium (modified with Hanks’ balanced salts) with 1, 5, or 10 μM of E2 or MT was added into the wells for the three treatment groups. In the control group, only M199 medium was added. Then, the pituitaries were incubated for 24 h. During incubation, pituitaries were collected at 6 h, 12 h, and 24 h, washed with fresh 1× PBS, and preserved at −80 °C, until tRNA extraction.
5
Soluble Leishmania Antigen Preparation
6
Soluble Leishmania Antigen Preparation
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Soluble Leishmania antigen (SLA) was prepared as previously described.63 Briefly, L. donovani amastigotes from clinical isolates (Kala‐azar Medical Research Center, Muzaffarpur, Bihar, India) were grown in Medium 199, Hanks’ Balanced Salts (M199; Thermo Fisher) until transformed into promastigotes, then cultured. 2–3 × 109 stationary‐phase promastigotes were harvested from culture and centrifuged at 3900 g for 20 min to obtain a parasite pellet, which was washed three times with cold 1 x PBS and resuspended in solution (10 mM Trizma hydrochloride solution (TRIS‐HCl; Sigma‐Aldrich), 1 mM pH 8.0 ethylenediaminetetraacetic acid (EDTA; Amresco, ELITechGroup, Puteaux, France), 1.6 mM phenylmethanesulphonyl fluoride (PMSF; HiMedia, Mumbai, India) and 50 mg mL‐1 N‐acetyl‐L‐leucyl‐Lleucyl‐ L‐argininal (leupeptin; Amresco)) at a concentration of 2–3 × 109 parasites m−1. The parasite suspension was sonicated 4–5 times for 15 s at 10 Hz and centrifuged at 2000 g for 30 min at 4°C. The lipid layer was removed from the surface of the supernatant, and the remaining solution was ultracentrifuged at 100 000 g for 4 h at 4°C. The supernatant was removed, dialysed against the PBS overnight and stored at –80°C until used. Protein concentration was measured using a Micro BCA Protein Assay Kit (Thermo Fisher), as per the manufacturer’s instructions.
7
Soluble Leishmania Antigen Preparation
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Soluble Leishmania antigen (SLA) was prepared as previously described (Gidwani et al., 2011 (link)). Briefly, L. donovani amastigotes from clinical isolates (Kala-azar Medical Research Center, Muzaffapur, Bihar, India) were grown in Medium 199, Hanks’ Balanced Salts (M199; Thermo Fisher) until transformed into promastigotes, then cultured. 2 × 109 stationary-phase promastigotes were harvested from culture and centrifuged at 3900 g for 20 minutes to obtain parasite pellet, which was washed three times with cold 1x PBS and resuspended in solution (10 mM Trizma® hydrochloride solution (TRIS-HCl; Sigma-Aldrich), 1 mM pH 8.0 ethylenediaminetetraacetic acid (EDTA; Amresco), 1.6 mM phenylmethanesulphonyl fluoride (PMSF; HiMedia, Mumbai, India), and 50 μg/ml N-acetyl-L-leucyl-L-leucyl-L-argininal (leupeptin; Amresco)) at a concentration of 2 × 109 parasites/ml. The parasite suspension was sonicated 4-5 times for 15 s at 10 Hz and centrifuged at 27,000 g for 30 minutes at 4°C. The lipid layer was removed from the surface of supernatant and the remaining solution was ultracentrifuged at 100,000 g for 4 hours at 4°C. The supernatant was removed, dialysed against the PBS overnight and stored at −80°C until use. Protein was measured using a Micro BCA Protein Assay Kit (Thermo Fisher) as per manufacturer’s instructions.
8
Isolation of Cochlear Coils from Rats
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Cochlear coils were isolated from male and female post-natal day 2–3 Sprague Dawley rats as previously described56 (link). Briefly, auditory bullae were removed and transferred into Medium 199 with Hank’s balanced salts (Life Technologies). The cartilaginous wall of the bulla was opened and the whole cochlea extracted. The stria vascularis and Reissner’s membrane were removed and the cochlea cut into three coils. The cochlear coils were placed onto Cell-Tak cell and tissue adhesive (BD Biosciences)-coated dishes (MatTek). For coating, cell adhesive was diluted to 70 μg ml−1 in 0.1 mM NaHCO3. The cochlear explants were incubated overnight in DMEM/F12 (Gibco), supplemented with 1% fetal bovine serum (Life Technologies) in a 37 °C, 5% CO2 incubator. The isolation was performed in accordance with the United Kingdom Animals (Scientific Procedures) Act of 1986 and in compliance with the Biological Services Management Group and the Biological Services Ethical Committee, University College London.
9
Isolation and Culture of Rat Cochlear Coils
10
Transient Transfection and Immunoprecipitation
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HEK293T cells were grown in DMEM with 10% FBS. Cells were transfected with plasmid DNA applying the calcium phosphate method and incubated for 24h. Cells were lysed in IP buffer (1% Triton-X 100, 20 mM Tris pH 7.5, 50 mM NaCl, 50 mM NaF, 15 mM Na4P2O7, 0.1 mM EDTA pH 8.0) supplemented with protease inhibitors. After centrifugation (43,000 rpm, 30 min, 4°C) the supernatants containing equal amounts of total protein were incubated with anti-FLAG M2 affinity beads (Sigma) for at least 1 h at 4°C. The precipitates were washed extensively with IP buffer and subjected to SDS-PAGE and immunoblotting analysis with anti-Flag and anti-V5 antibodies (Sigma).
For the analysis of S6K phosphorylation HEK293T cells were transfected with epitope-tagged Rheb and Dbl protein. 24h after transfection, cells were starved 1h in MEM, Hank’s Balanced Salts (from life technologies) and then lysed in IP buffer. Lysates were centrifuged (43,000 rpm, 30 min, 4°C). Equal amounts of protein samples were analyzed by immunoblotting. Anti-p70-S6K and anti-phospho-p70-S6K antibodies (Thr389) were obtained from Cell signaling, anti-actin antibodies from Sigma.
Expression constructs are described in detail in theSupplementary Table 5 .
For the analysis of S6K phosphorylation HEK293T cells were transfected with epitope-tagged Rheb and Dbl protein. 24h after transfection, cells were starved 1h in MEM, Hank’s Balanced Salts (from life technologies) and then lysed in IP buffer. Lysates were centrifuged (43,000 rpm, 30 min, 4°C). Equal amounts of protein samples were analyzed by immunoblotting. Anti-p70-S6K and anti-phospho-p70-S6K antibodies (Thr389) were obtained from Cell signaling, anti-actin antibodies from Sigma.
Expression constructs are described in detail in the
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