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Isovitalex

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IsoVitaleX is a sterile, cell culture medium supplement manufactured by BD. It is designed to support the growth and maintenance of various cell lines in vitro. The product provides a source of essential nutrients, growth factors, and other components required for cell culture applications.

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Difco gc medium base agar, by BD (9 mentions) Gc agar, by BD (6 mentions) Bovine hemoglobin, by BD (5 mentions) Mueller hinton broth (mhb), by BD (5 mentions) Hemoglobin, by BD (4 mentions) Mh broth, by BD (4 mentions) Defibrinated horse sterile blood, by Thermo Fisher Scientific (3 mentions) Campy pak jar, by Thermo Fisher Scientific (3 mentions) Protease peptone, by Thermo Fisher Scientific (3 mentions) Brucella broth, by BD (3 mentions) Phosphate buffered saline (pbs), by Corning (3 mentions) Vancomycin, by Merck Group (3 mentions) Trimethoprim, by Merck Group (3 mentions) Horse serum, by Thermo Fisher Scientific (3 mentions) Mueller hinton agar (mha), by BD (3 mentions) Mh chocolate agar plates, by BD (3 mentions) Haemoglobin, by BD (3 mentions) Gc agar plates, by Thermo Fisher Scientific (2 mentions) Bl21 de3, by Thermo Fisher Scientific (2 mentions) Agar, by BD (2 mentions)

123 protocols using isovitalex

1

Culturing Francisella tularensis Strains

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Bacterial strains used in this study are listed in Table 1. F. tularensis species were routinely cultured on enriched chocolate agar plates (Remel) at 37°C. For liquid culture, either brain heart infusion broth (BHI) supplemented with 1% IsoVitaleX (Becton-Dickinson), modified Muller-Hinton (MMH) supplemented with 2% IsoVitaleX, or Chamberlain’s Defined Medium (CDM) (Chamberlain, 1965 (link)) was used. In some instances, BHI and MMH were pH adjusted as indicated.
Mlynek K.D., Lopez C.T., Fetterer D.P., Williams J.A, & Bozue J.A. (2022). Phase Variation of LPS and Capsule Is Responsible for Stochastic Biofilm Formation in Francisella tularensis. Frontiers in Cellular and Infection Microbiology, 11, 808550.
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2

Antimicrobial Susceptibility of Neisseria

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To determine the susceptibility concentration, Neisseria meningitidis H44/76 or Neisseria gonorrhoeae MS11 was grown as a lawn on GC-agar (BD Biosciences) with 1% IsoVitaleX (BD Biosciences) at 37 °C in a moist atmosphere with 5% CO2 for 16 to 18 h. Cells from the agar-media plates were collected with a sterile cotton swab and resuspended in Brain Heart Infusion (BHI, from Difco) growth media with 1% IsoVitaleX (Becton Dickinson, Sparks, USA) to about 106 CFU/mL. Each compound was diluted in growth media to 2× working concentration. 50 μL of the cell suspension was mixed with 50 μL of the diluted compound in a single well of a 96-well plate. These plates were sealed with Breathe-Easy sealing membrane (Sigma-Aldrich) and were then incubated at 37 °C in a moist atmosphere with 5% CO2. After 16 to 18 h, minimum inhibitory concentrations were determined optically as the lowest concentration of compound that completely inhibited growth. Treated cultures were then serially diluted in PBS+2 (link) (PBS supplemented with MgCl2 and CaCl2) and spotted on GC agar plates supplemented with 1% IsoVitaleX and incubated for 18–20 h, following which, CFUs were counted. Treatment concentrations resulting in hazy or no growth were considered as inhibitory concentrations. For analysis, the percentage of CFUs recovered post-treatment to pretreatment are reported.
Binepal G., Mabanglo M.F., Goodreid J.D., Leung E., Barghash M.M., Wong K.S., Lin F., Cossette M., Bansagi J., Song B., Serrão V.H., Pai E.F., Batey R.A., Gray-Owen S.D, & Houry W.A. (2020). Development of Antibiotics That Dysregulate the Neisserial ClpP Protease. ACS infectious diseases, 6(12), 3224-3236.
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3

Francisella tularensis Strain Maintenance

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All bacterial strains, plasmids, and primers used in this study are listed in Additional file 6: Table S1. F. tularensis subsp. holarctica LVS was obtained from Ft. Collins, CO. Francisella strains were maintained on chocolate agar supplemented with 1% IsoVitalex (BD), Chamberlains defined media (CDM) [25 (link)], or in BHI supplemented with 1% IsoVitalex at 37°C.
Miller C.N., Steele S.P., Brunton J.C., Jenkins R.J., LoVullo E.D., Taft-Benz S.A., Romanchuk A., Jones C.D., Dotson G.D., Collins E.J, & Kawula T.H. (2014). Extragenic suppressor mutations in ΔripA disrupt stability and function of LpxA. BMC Microbiology, 14, 336.
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4

Cultivation of Francisella tularensis

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The bacterial strains used in this study are listed in Table 1. The F. tularensis subsp. holarctica live vaccine strain (LVS) (ATCC 29684; American Type Culture Collection, Rockville, MD) was obtained from BEI Resources, Manassas, VA. F. tularensis cultures were grown on Mueller Hinton (MH) chocolate agar plates (BD Biosciences, San Jose, CA) or modified MH-chocolate agar (MMH) (Bakshi et al., 2006 (link)) supplemented with IsoVitaleX at 37°C with 5% CO2 or in MH broth (BD Biosciences, San Jose, CA) supplemented with ferric pyrophosphate and IsoVitaleX (BD Biosciences, San Jose, CA) at 37°C with shaking (160 rpm). Active mid-log-phase bacteria grown in MH broth were harvested and stored at −80°C. The Escherichia coli DH5-α strain was used for cloning experiments. E. coli cultures were grown in Luria-Bertani (LB) broth or on LB agar plates. When necessary, kanamycin (25 µg/mL) or hygromycin (200 µg/mL) was included in the broth and agar media for the selection of transformants, mutants or transcomplemented strains.
Ma Z., Russo V.C., Rabadi S.M., Jen Y., Catlett S.V., Bakshi C.S, & Malik M. (2016). Elucidation of a Mechanism of Oxidative Stress Regulation in Francisella tularensis Live Vaccine Strain. Molecular microbiology, 101(5), 856-878.
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5

Culturing Francisella and Salmonella Strains

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Francisella tularensis Live Vaccine Strain (F. tularensis LVS) and Salmonella Typhimurium strains were obtained from BEI Resources Manassas, VA. The Francisella cultures were grown on Mueller-Hinton (MH) chocolate agar plates (BD Biosciences, San Jose, CA) supplemented with IsoVitaleX at 37°C with 5% CO2 or in MH broth (BD Biosciences, San Jose, CA) supplemented with ferric pyrophosphate and IsoVitaleX (BD Biosciences, San Jose, CA) at 37°C with shaking (160rpm). Luria-Bertani broth or McConkeys agar was used for culturing S. Typhimurium. All bacterial culture stocks grown to the mid-log phase were stored at −80°C and aliquots were thawed for use in experiments. All bacteria were handled in a Bio-Safety Level-2 cabinet.
Suresh R.V., Bradley E.W., Higgs M., Russo V.C., Alqahtani M., Huang W., Bakshi C.S, & Malik M. (2021). Nlrp3 Increases the Host’s Susceptibility to Tularemia. Frontiers in Microbiology, 12, 725572.
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6

Culturing Francisella tularensis and Escherichia coli

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The bacterial strains, plasmids, and primers used in this study are listed in Table S1. The F. tularensis subsp. holarctica live vaccine strain (F. tularensis LVS) (ATCC 29684; American Type Culture Collection, Rockville, MD) was used in this study. The F. tularensis cultures were grown on Mueller-Hinton (MH) chocolate agar plates (BD Biosciences, San Jose, CA) supplemented with IsoVitaleX at 37°C with 5% CO2; MH-broth (BD Biosciences, San Jose, CA) supplemented with ferric pyrophosphate and IsoVitaleX (BD Biosciences, San Jose, CA); or in Brain-Heart Infusion (BHI)-broth at 37°C with shaking (160 rpm). Active mid-log phase bacteria grown in MH- or BHI broth were harvested and stored at −80°C; 1ml aliquots were thawed periodically for use. Escherichia coli strain DH5α was used for routine cloning. E. coli cultures were grown in Luria-Bertani (LB) broth or on LB agar plates. When necessary, kanamycin (10 μg/ml), or hygromycin (100 μg/ml) was included in broth and agar media for selection purposes.
Ma Z., Banik S., Rane H., Mora V.T., Rabadi S.M., Doyle C.R., Thanassi D.G., Bakshi C.S, & Malik M. (2014). EmrA1 Membrane Fusion Protein of Francisella tularensis LVS is required for Resistance to Oxidative Stress, Intramacrophage Survival and Virulence in Mice. Molecular microbiology, 91(5), 976-995.
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7

Neisseria gonorrhoeae Infection Protocols

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Neisseria gonorrhoeae F62 was isolated from an uncomplicated infection (Kellogg, Peacock, Deacon, Brown, & Pirkel, 1963). Neisseria gonorrhoeae 15253 was isolated from a disseminated infection (O'Brien, Goldenberg, & Rice, 1983). Both strains were piliated. The mutant strains of F62 (ΔlgtD, ΔlgtA, ΔlgtE, and ΔlgtF) were constructed using plasmids and methods described previously (Gulati et al., 2015). The LOS phenotype of the mutants was verified by LOS staining of protease K‐treated whole‐cell samples that were separated on a 12% Bis–Tris gel with MES running buffer. N. gonorrhoeae were grown overnight on chocolate agar plates with IsoVitaleX (BD Bioscience) or in GC broth supplemented with IsoVitaleX at 37°C and 5% CO2. When indicated, growth media was supplemented with 30 µM CMP‐Neu5Ac (Nacalai USA, Inc.). Incorporation of Neu5Ac has been confirmed by loss of Erythrina Cristagalli Lectin (ECA, Vector Laboratories), which binds to the lactosamine epitope. For all binding and infection studies, bacteria were cultivated to an optical density at 600 nm equivalent to 0.4–0.6. THP‐1 cells were grown in RPMI‐1640 (Gibco) with 10% fetal calf serum (Gemini Bio‐Products) at 37°C and 5% CO2.
Landig C.S., Hazel A., Kellman B.P., Fong J.J., Schwarz F., Agarwal S., Varki N., Massari P., Lewis N.E., Ram S, & Varki A. (2019). Evolution of the exclusively human pathogen Neisseria gonorrhoeae: Human‐specific engagement of immunoregulatory Siglecs. Evolutionary Applications, 12(2), 337-349.
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8

Culturing Gonococcal Strain 1291

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Gonococcal strain 1291 (male gonococcal urethritis isolate, provided by M. A. Apicella) was grown at 37°C with 5% CO2 on GC agar plates (Oxoid) supplemented with 1% IsoVitalex (Becton, Dickinson) for ∼16 h. The majority of the gonococcal population investigated expressed pili and Opa, as determined by phase-contrast microscopy. For experiments with Opa-negative N. gonorrhoeae (Table 3), clear colonies were selected by phase-contrast microscopy.
Semchenko E.A., Everest-Dass A.V., Jen F.E., Mubaiwa T.D., Day C.J, & Seib K.L. (2019). Glycointeractome of Neisseria gonorrhoeae: Identification of Host Glycans Targeted by the Gonococcus To Facilitate Adherence to Cervical and Urethral Epithelial Cells. mBio, 10(4), e01339-19.
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9

Francisella tularensis LVS Strain Generation

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The live vaccine strain of Francisella tularensis subspecies holartica (LVS) was obtained from the CDC (Atlanta, GA). The LVS clpB (FTL_0094) deletion strain was generated as previously described (16 (link)). Bacteria were grown at 37°C on chocolate agar supplemented with 1% IsoVitalex (Becton-Dickinson). To prepare bacterial inoculations, bacteria were removed from a lawn grown on chocolate agar and resuspended in sterile PBS at an OD600=1 (equivalent to 1×1010 CFU/mL). The number of viable bacteria was determined by serial dilution and plating on chocolate agar.
Roberts L.M., Davies J.S., Sempowski G.D, & Frelinger J.A. (2014). IFN-γ, but not IL-17A, is required for survival during secondary pulmonary Francisella tularensis Live Vaccine Stain infection. Vaccine, 32(29), 3595-3603.
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10

Bacterial Vesicle Production Protocols

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Lactobacillus reuteri DSM 17938 [42 (link)], BioGaia AB, Stockholm, Sweden], and Helicobacter pylori ATCC 43629, were used for this study. The choice of these two microorganisms linked to the fact that in previous works we have demonstrated that they produce vesicles containing DNA, which are released both from the planktonic and the biofilm phenotypes [15 (link),16 (link)]. L. reuteri DSM 17938 was spread on deMan, Rogosa, Sharpe Agar (MRS) (Oxoid Limited, Hampshire, UK), and incubated at 37 °C for 24 h in an anaerobic atmosphere (Anaerogen Pak Jar, Oxoid Ltd., Basingstoke, UK), while H. pylori ATCC43629 was plated on Chocolate Agar (Oxoid Ltd.) supplemented with 1% (v/v) of IsoVitaleX (Becton Dickinson, Franklin Lakes, NewJersey, NJ, USA) and 10% (v/v) of defibrinated horse sterile blood (Oxoid Ltd.), and finally incubated at 37 °C for 3 days in a microaerophilic atmosphere (Campy PakJar; Oxoid Ltd.).
Puca V., Ercolino E., Celia C., Bologna G., Di Marzio L., Mincione G., Marchisio M., Miscia S., Muraro R., Lanuti P, & Grande R. (2019). Detection and Quantification of eDNA-Associated Bacterial Membrane Vesicles by Flow Cytometry. International Journal of Molecular Sciences, 20(21), 5307.
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