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Middlebrook oadc

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Middlebrook OADC is a nutritional supplement used in the cultivation of mycobacterial species, such as Mycobacterium tuberculosis, the causative agent of tuberculosis. It provides essential nutrients and growth factors to support the growth of these slow-growing bacteria in laboratory settings.

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Lab products found in correlation

M tuberculosis, by BD (1 mentions) Calcium chloride dihydrate, by Merck Group (1 mentions) Soy phosphatidylcholine, by Lipoid (1 mentions) Neutral red, by Merck Group (1 mentions) Oleylamine, by Merck Group (1 mentions) Rifampicin, by Merck Group (1 mentions) Sodium chloride (nacl), by Merck Group (1 mentions) Potassium chloride (kcl), by Merck Group (1 mentions) Dopalen, by Ceva (1 mentions)

7 protocols using middlebrook oadc

1

Infection of Macrophages with Fluorescent M. tuberculosis

Cited 1 time
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M. tuberculosis was grown to log phase in 7H9 liquid media (BD 271310) supplemented with Middlebrook OADC (Sigma M0678), 0.5% glycerol, 0.05% Tween-80 in roller bottles at 37°C. M. tuberculosis Erdman strain expressing eGFP under control of the MOP promoter was a gift from Dr. Sarah Stanley’s laboratory. Macrophages were infected with fluorescent M. tuberculosis using a modified version of the spinfection protocol as previously described (Watson et al., 2015 (link)). Mycobacteria were washed in PBS three times and directly inoculated into the macrophage tissue culture wells at an MOI of 10. Following centrifugation, infected cells were incubated at 37°C for one day post-infection. The wells were washed with PBS and fixed with 4% PFA before staining for microscopy.
Hiatt J., Cavero D.A., McGregor M.J., Zheng W., Budzik J.M., Roth T.L., Haas K.M., Wu D., Rathore U., Meyer-Franke A., Bouzidi M.S., Shifrut E., Lee Y., Kumar V.E., Dang E.V., Gordon D.E., Wojcechowskyj J.A., Hultquist J.F., Fontaine K.A., Pillai S.K., Cox J.S., Ernst J.D., Krogan N.J, & Marson A. (2021). Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins. Cell reports, 35(6), 109105.
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2

Detailed Cultivation and Infection Protocol for Mycobacterium tuberculosis

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All Mtb experiments were performed with Mtb Erdman strain or strains derived from Mtb Erdman strain. Low passage frozen stocks of Mtb were grown to log phase in 7H9 (BD) liquid media supplemented with 10% Middlebrook OADC (Sigma), 0.05% Tween-80 and 0.5% glycerol in roller bottles at 37°C. Mtb-LUX expressing luciferase from the luxCDABE operon has been described previously and was cultured as described above [36 (link)]. WT or ΔeccC Mtb expressing eGFP under control of the MOP promoter was a gift from Dr. Sarah Stanley’s laboratory [63 (link)], and was cultured as described above. For BMM infections, log phase bacteria were washed twice with PBS and sonicated three times at 90% amplitude for 30 seconds each then diluted into BMM media (DMEM with 10% FBS, 2 mM L-glutamine, 10% MCSF, 11 mg/mL sodium pyruvate) prior to infection. For THP-1 and RAW 264.7 infections, 10 mL of the Mtb culture was washed twice in PBS with 10% heat-inactivated horse serum. After washing, Mtb was resuspended in 5 mL PBS with 10% heat-inactivated horse serum and sonicated twice at 50% amplitude for 15 seconds. Mtb was diluted into DMEM with 10% heat-inactivated horse serum, and this inoculum was added to THP-1 cells prior to spinfection.
Morrison H.M., Craft J., Rivera-Lugo R., Johnson J.R., Golovkine G.R., Bell S.L., Dodd C.E., Van Dis E., Beatty W.L., Margolis S.R., Repasy T., Shaker I., Lee A.Y., Vance R.E., Stanley S.A., Watson R.O., Krogan N.J., Portnoy D.A., Penn B.H, & Cox J.S. (2023). Deficiency in Galectin-3, -8, and -9 impairs immunity to chronic Mycobacterium tuberculosis infection but not acute infection with multiple intracellular pathogens. PLOS Pathogens, 19(6), e1011088.
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3

Culturing M. tuberculosis for Macrophage Infections

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M. tuberculosis (Erdman) was used for macrophage infections. M. tuberculosis was grown to log phase in 7H9 liquid media (BD) supplemented with 10% Middlebrook OADC (Sigma), 0.5% glycerol, 0.05% Tween80 in roller bottles at 37°C.
Budzik J.M., Swaney D.L., Jimenez-Morales D., Johnson J.R., Garelis N.E., Repasy T., Roberts A.W., Popov L.M., Parry T.J., Pratt D., Ideker T., Krogan N.J, & Cox J.S. (2020). Dynamic post-translational modification profiling of Mycobacterium tuberculosis-infected primary macrophages. eLife, 9, e51461.
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4

Characterization of Mycobacterium tuberculosis Mutants

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All Mtb strains were cultured in 7H9 liquid media (BD) supplemented with 10% Middlebrook OADC (Sigma), 0.5% glycerol, 0.05% Tween80 in roller bottles at 37°C. Transposon mutants were obtained from ATCC and carry Himar1 transposon insertions with the Kanamycin resistance gene in the indicated locus. Transposon insertion sites were validated by PCR and Sanger sequencing (Lamichhane et al., 2003 (link)). For genetic complementation studies, the predicted promoter region and open reading frame were cloned into the SacI site of the integrating vector pBU (Vultos et al., 2006 (link)) which had been modified to confer Zeocin resistance, and strains were selected with 25μg/ml Zeocin. Control strains carried pBU with a ~100 nucleotide fragment of GFP as unique sequence tag (see Key Resources Table). For luminescent growth assays, strains carried the containing a codon-optimized luxBCADE operon expressed from a MOPS promoter carried on the pmv306-Hyg integrating vector (Craney et al., 2007 (link)). The ΔeccC strain was made by homologous recombination using the pMSG361 vector (Rosenberg et al., 2015 (link)).
Penn B.H., Netter Z., Johnson J.R., Von Dollen J., Jang G.M., Johnson T., Ohol Y.M., Maher C., Bell S.L., Geiger K., Golovkine G., Du X., Choi A., Parry T., Mohapatra B.C., Storck M.D., Band H., Chen C., Jäger S., Shales M., Portnoy D.A., Hernandez R., Coscoy L., Cox J.S, & Krogan N.J. (2018). An Mtb-Human Protein-Protein Interaction Map Identifies a Switch Between Host Anti-Viral and Anti-Bacterial Responses. Molecular cell, 71(4), 637-648.e5.
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5

Infection of Macrophages with Fluorescent M. tuberculosis

Cited 1 time
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M. tuberculosis was grown to log phase in 7H9 liquid media (BD 271310) supplemented with Middlebrook OADC (Sigma M0678), 0.5% glycerol, 0.05% Tween-80 in roller bottles at 37°C. M. tuberculosis Erdman strain expressing eGFP under control of the MOP promoter was a gift from Dr. Sarah Stanley’s laboratory. Macrophages were infected with fluorescent M. tuberculosis using a modified version of the spinfection protocol as previously described (Watson et al., 2015 (link)). Mycobacteria were washed in PBS three times and directly inoculated into the macrophage tissue culture wells at an MOI of 10. Following centrifugation, infected cells were incubated at 37°C for one day post-infection. The wells were washed with PBS and fixed with 4% PFA before staining for microscopy.
Hiatt J., Cavero D.A., McGregor M.J., Zheng W., Budzik J.M., Roth T.L., Haas K.M., Wu D., Rathore U., Meyer-Franke A., Bouzidi M.S., Shifrut E., Lee Y., Kumar V.E., Dang E.V., Gordon D.E., Wojcechowskyj J.A., Hultquist J.F., Fontaine K.A., Pillai S.K., Cox J.S., Ernst J.D., Krogan N.J, & Marson A. (2021). Efficient generation of isogenic primary human myeloid cells using CRISPR-Cas9 ribonucleoproteins. Cell reports, 35(6), 109105.
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6

Mycobacterial Growth Inhibition Assay

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Rifampicin, purity ≥ 97% (Sigma Aldrich, St. Louis, MO, USA); Heparin, 5000 U.I./mL, (BlauFarmacêutica®, Cotia, Brazil); Dopalen, ketamine 10% injectable, veterinary use (Ceva, Paulínia, Brazil); Calmiun, xylazine 2% injectable, veterinary use (Agener União Saúde Animal, São Paulo, Brazil); Bacter Bacterial agar (Becton Dickinson, East Rutherford, EUA); Cetylalcoholethoxylated 20 OE and propoxylated 5 OP-Procetyl AWS (Croda, Campinas, Brazil); Calcium chloride dihydrate (Merck, St. Louis, CO, USA); Potassium chloride (Merck); Sodium Chloride (Merck); soy phosphatidylcholine (Lipoid GMBH, Geneva, Switzerland); culture medium-Dulbecco’s Modified Eagle Medium (Hyclone, Logan, UT, USA); Brook Middlebrook culture medium-7H11 (Difcot, St. Louis, CO, USA); Oleylamine (Sigma Aldrich, St. Louis, CO, USA); Middlebrook-OADC (Sigma Aldrich, St. Louis, MO, USA); Neutral Red (Sigma Aldrich, St. Louis, MO, USA).
Santos K.P., Rodero C.F., Ribeiro C.M., Gremião M.P., Peccinini R.G., Pavan F.R., Pearce C., Gonzalez-Juarrero M, & Chorilli M. (2022). Development of a Mucoadhesive Liquid Crystal System for the Administration of Rifampicin Applicable in Tuberculosis Therapy. Life, 12(8), 1138.
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7

Fluorescent Mycobacterium Infection Assay

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M. tuberculosis was grown to log phase in 7H9 liquid media (BD 271310) supplemented with Middlebrook OADC (Sigma M0678), 0.5% glycerol, 0.05% Tween-80 in roller bottles at 37ºC. M. tuberculosis Erdman strain expressing eGFP under control of the MOP promoter was a gift from Dr. Sarah Stanley's laboratory. Macrophages were infected with fluorescent M. tuberculosis using a modified version of the spinfection protocol as previously described (Watson et al., 2015) . Mycobacteria were washed in PBS three times and directly inoculated into the macrophage tissue culture wells at an MOI of 10. Following centrifugation, infected cells were incubated at 37°C for one day post-infection. The wells were washed with PBS and fixed with 4% PFA before staining for microscopy.
Hiatt J., Cavero D.A., McGregor M., Gordon D.E., Zheng W., Budzik J.M., Roth T.L., Haas K.M., Rathore U., Meyer‐Franke A., Bouzidi M.S., Hultquist J.F., Wojcechowskyj J.A., Fontaine K.A., Pillai S.K., Cox J.S., Ernst J., Krogan N.J., & Marson A. (2020). Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins.
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