Whole-Blood Stimulation Assay for Mycobacterial Immunity
Whole-blood samples from one healthy control, one healthy travel control, P1 and P2 were collected in heparin-containing collection tubes. Samples were diluted 1:2 in RPMI 1640 supplemented with 100 IU/mL penicillin and 100 μg/mL streptomycin (Thermo Fisher Scientific). Stimulation was performed in a 48-well plate. Briefly, 1 mL of whole-blood was used per well and per set of conditions. Samples were incubated with medium alone, with live BCG (M. bovis-BCG, Pasteur substrain) at a MOI of 20, or with BCG plus IL-12 (20 ng/mL; #219-IL, R&D Systems), or BCG plus IFN-γ (Imukin, Boehringer Ingelheim) for 48 hours at 37°C under an atmosphere containing 5% CO2. The supernatants were collected and used for ELISA. Data were compared to published findings for healthy and travel controls.61 (link)
Neehus A.L., Carey B., Landekic M., Panikulam P., Deutsch G., Ogishi M., Arango-Franco C.A., Philippot Q., Modaresi M., Mohammadzadeh I., Berndt M.C., Rinchai D., Le Voyer T., Rosain J., Momenilandi M., Martin-Fernandez M., Khan T., Bohlen J., Han J.E., Deslys A., Bernard M., Gajardo-Carrasco T., Soudée C., Le Floc’h C., Migaud M., Seeleuthner Y., Jang M.S., Nikolouli E., Seyedpour S., Begueret H., Emile J.F., Le Guen P., Tavazzi G., Julia Colombo C.N., Marzani F.C., Angelini M., Trespidi F., Ghirardello S., Alipour N., Molitor A., Carapito R., Mazloomrezaei M., Rokni-Zadeh H., Changi-Ashtiani M., Brouzes C., Vargas P., Borghesi A., Lachmann N., Bahram S., Crestani B., Pahari S., Schlesinger L.S., Marr N., Bugonovic D., Boisson-Dupuis S., Béziat V., Abel L., Borie R., Young L.R., Deterding R., Shahrooei M., Rezaei N., Parvaneh N., Craven D., Gros P., Malo D., Sepulveda F.E., Nogee L.M., Aladjidi N., Trapnell B.C., Casanova J.L, & Bustamante J. (2023). Human inherited CCR2 deficiency underlies progressive polycystic lung disease. Cell, 187(2), 390-408.e23.
Corresponding Organization : Howard Hughes Medical Institute
Other organizations :
McGill University, University of Washington, Universidad de Antioquia, Tehran University of Medical Sciences, Children's Medical Center, University of Tehran, Babol University of Medical Sciences, Universal Scientific Education and Research Network, Icahn School of Medicine at Mount Sinai, Child Health and Development Institute, Jackson Laboratory, Centre National de la Recherche Scientifique, Délégation Paris 5, Université Paris Sciences et Lettres, Institut Pierre-Gilles de Gennes pour la Microfluidique, Medizinische Hochschule Hannover, Hôpital Cardiologique du Haut-Lévêque, Hôpital Ambroise-Paré, University of Pavia, Université de Strasbourg, Hôpital Civil, Strasbourg, Zanjan University of Medical Sciences, Institute for Research in Fundamental Sciences, École Polytechnique Fédérale de Lausanne, Texas Biomedical Research Institute, Hamad bin Khalifa University, Children's Hospital of Philadelphia, Children's Hospital Colorado, University of Colorado Denver, KU Leuven, Rainbow Babies & Children's Hospital, Johns Hopkins Medicine, Johns Hopkins University
Stimulation conditions: 1) medium alone, 2) live BCG (M. bovis-BCG, Pasteur substrain) at a MOI of 20, 3) BCG plus IL-12 (20 ng/mL), 4) BCG plus IFN-γ
dependent variables
Cytokine/protein levels in the supernatants (measured by ELISA)
control variables
Whole-blood samples from one healthy control and one healthy travel control
Samples diluted 1:2 in RPMI 1640 supplemented with 100 IU/mL penicillin and 100 μg/mL streptomycin
Incubation time of 48 hours at 37°C under 5% CO2 atmosphere
controls
Positive control: Whole-blood samples from one healthy control
Negative control: Whole-blood samples from one healthy travel control
Annotations
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