The antibiotic used was Gentamicin Sulfate (MP Biomedical, Cat No. 1676045, Santa Ana, CA, USA), an aminoglycoside that interrupts protein synthesis by binding to the 30S subunit of the bacterial ribosome and which was previously flown in experiments onboard STS-69 and STS-73 [67 ] and the Soviet/Russian space station Mir [68 ] (reported in the last reference simply as Gentamicin, with no further details). Gentamicin Sulfate solutions were prepared in distilled water and filter-sterilized (0.20 μm) for flight. Their concentrations varied so that, when introduced into the culture, they would range from 25 to . They were stored at 4°C until needed for loading the FPAs (see Hardware, Sample Preparation and Loading, and Operations Timeline).
Gentamicin sulfate
Manufactured by MP Biomedicals
Sourced in United States, Germany, Macao
Gentamicin Sulfate is a broad-spectrum antibiotic used in various laboratory applications. It is a water-soluble powder that inhibits bacterial protein synthesis, making it effective against a range of Gram-positive and Gram-negative bacteria. Gentamicin Sulfate is commonly used in cell culture media to prevent bacterial contamination.
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4 protocols using gentamicin sulfate
1
Antibiotic Experiments in Microgravity
2
Induction of Mixed Chimerism in Mice
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C57BL/6 recipient mice received 20 × 106 unseparated BALB/c, B10.D2, CB6F1, or F1.BALB/c BM cells (d0). CD45.1 recipient mice were transplanted with 20 × 106 unseparated BALB.B BM cells (d0). BM cells were collected from long and hip bones and preserved in M199 medium (Sigma‐Aldrich, St. Louis, MO) supplemented with 4 μg/mL Gentamicin Sulfate (MP Biomedicals, Irvine, CA) and 10 mM Hepes Buffer (MP Biomedicals). All BMT recipients additionally received CB consisting of α‐CD40L (1 mg: d0; clone MR1; Bio X Cell, West Lebanon, NH) and CTLA4‐Ig (0.5 mg: d2; Bristol‐Myers Squibb, New York, NY) and a short course of rapamycin (0.1 mg: d–1, d0, d2; LC Laboratories, Woburn, MA). Selected recipients of BALB/c, BALB.B, or B10.D2 BM additionally received α‐NK1.1 (0.25 mg: clone PK136; Bio X Cell) either at the time of transplantation (d–1, d2, d5, d8, short‐α‐NK1.1) or regularly until the end of follow‐up (d–1, d2, d5, d8, d28, d56, d84, 112, 140, 168) (long‐α‐NK1.1). Antibodies, fusion proteins, and rapamycin were administered intraperitoneally (i.p.). Mixed chimerism was defined as having at least 2 lineages displaying >0.5% donor cells.
3
In Vitro Maturation of Oocytes
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In order to obtain MII oocytes from germinal vesicle-stage oocytes via in vitro maturation (IVM), germinal vesicle-stage oocytes were retrieved from the ovary 48 h after eCG by puncturing its surface in HCZB. Oocytes were deprived of surrounding cumulus cells by gentle pipetting and incubated for 20 min at 37 °C in HCZB with the addition of 0.05 μg/mL Hoechst. Fluorescent imaging was performed on the stage of an inverted microscope using a 20X S-Fluor objective. The oocytes were sorted into two groups according to the distribution of chromatin around the nucleolus (SN or NSN;60 (link)) and allowed to mature overnight in α-MEM (cat. no. M4526, Sigma-Aldrich Chemie GmbH, Germany) supplemented with 10% fetal bovine serum and 50 I.U./mL gentamicin sulfate (MP Biomedicals). The SN-MII and NSN-MII oocytes were imaged side by side using a 10X objective, as described for the (super)ovulated oocytes.
4
Allogeneic Bone Marrow Transplant with Costimulation Blockade
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Groups of age-matched C57BL/6 recipients received costimulation blockade consisting of α-CD40L (CD154) mAb (MR1, 1 mg, d0) (BioXcell) with or without CTLA4-Ig (0.5 mg, d2) (Bristol Myers Squibb Pharmaceuticals, Princeton, NJ) and 20×106 unseparated BM cells recovered from naïve BALB/c donors (d0). BM cells were collected in M199 medium (Sigma-Aldrich, St. Louis, MO) supplemented with 4μg/ml Gentamicin Sulfate (MP Biomedicals, Santa Ana, USA) and 10mM Hepes Buffer (MP Biomedicals) (BM medium). Selected groups were administered with a short course of rapamycin (0.1 mg, d-1, d0 and d2) (LC Laboratories, Woburn MA) and either received in vitro activated or IL-2 complex expanded Treg populations (3×106) at the time of BMT or IL-2 complexes (d-4, -3 and -2; 5μg IL-2 / 25μg α-IL-2). Indicated groups of recipients were irradiated with 1Gy total body irradiation (TBI) prior to BMT (d-1) using a Xylon X-Ray unit [25 (link)]. Irradiated mice were additionally treated with or without IL-2 complexes (d3, d5; 1μg IL-2 / 5μg α-IL-2). All reagents were administered i.p. in a phosphate buffered solution (PBS) and BM cells were injected intravenously (i.v.) in BM medium.
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