This in vitro assay measures mycobacterial metabolic activity in whole blood to evaluate the effect of interactions between mycobacteria and host immune cells. First, add reported gene tagged mycobacteria to whole human blood in order to provide a real-time readout over a time course. Collect sample supernatants for cytokine measurements and cell palettes for RNA analyses continue to lies the red blood cells and using serial dilutions and bioluminescence measure metabolic activity of the mycobacteria.
Finally, calculator ratio between the final bioluminescence signal expresses relative light units and the bioluminescence signal at the time of inoculation. The results indicate the host's ability to contain mycobacteria. This whole blood reported gene assay can help answer key questions in the field.
For instance, does the presence of certain effector immune cells in a blood sample have a functional impact on the organisms in this case? Microbacteria.The main advantage of this technique over existing methods is that the enumeration of mycobacteria can be obtained in real time rather than counting colony forming units after three weeks of incubation and exact and reproducible inoculum can be determined each time the experiment is conducted. First, grow A BCG luxe culture to mid logarithmic phase using the luminometer tubes.
Prepare duplicate samples of one milliliter of a one to 10 dilution of BCG luxe culture in sterile PBS at the luminometer. Load the YL outer Hyde substrate tube and prime the injector. Then load the sample tubes and take bioluminescence measurements.
Adopt optimal culture density. Add an equal volume of sterile 30%glycerol to the culture and gently mix store 1.5 milliliter aliquots of minus 80 degrees Celsius. In order to determine the R-L-U-C-F-U correlations of the stock preparation, inoculate a 15 milliliter culture with one third aliquot of BCG Lux and amplify at 37 degrees Celsius for four days.
On each day, make serial tenfold dilutions for colony forming unit determination. Also set up the same dilution in parallel and duplicate the luminescence measurements now spread 100 microliters of each dilution onto plates of Middlebrook. Seven H 11 agar.
Seal each plate with biofilm and incubate in a plastic bag at 37 degrees Celsius for the next four weeks. Inspect the plates regularly for appearance of colonies and enumerate colony forming units on a colony counter counting back to the dilution you've plated out. Determine the R-L-U-C-F-U ratio of the stock culture as specified in the accompanying manuscript for uses inoculation into whole blood.
Dilute the BCG luxe culture in logarithmic growth in PBS. Now transfer three to five milliliters of venous blood from a tube containing preservative-free heparin to a 30 milliliter sterin tube and dilute with an equal volume of supplemented RPMI medium without antibiotics. Aliquot 900 microliters into sterile B tubes for triplicate measurements at each time point for the growth controls aliquot.
900 microliters of supplemented middle Brooks, seven H nine in duplicates per time point. Continue to add 100 microliters of the diluted BCG luxe to each sample. Mix well and incubate samples on a rucker at define time points.
Centrifuge the samples at 2000 times gravity for 10 minutes. Carefully aliquot 300 microliters of super natum for subsequent cytokine analysis. After adding 300 microliters of PBS to each sample, aspirate the contents into 30 milliliters sterile in tubes.
Then rinse each biju tube with two milliliters of distilled water. Then add eight milliliters of distilled water to the sterin tube. Vortex the biju tube for five seconds and tip its contents into the corresponding sterin tube and incubate for 10 minutes.
Centrif, use the samples and decant sate into disinfectant. Add a few glass beads to each pallet and cortex. Then add one milliliter of sterile PBS and vortex again.
Next in luminometer tubes, make one to 10 dilution in PBS for each sample in duplicate. Finally, determine the luciferase activity in these whole blood lax assays, it is important to use bacteria in logarithmic phase of growth. This typical growth curve of BCG luxe over 96 hours indicates doubling time is about 24 hours until stationary phase is reached here.
Growth ratios were calculated for three different adult donors from luminometer readings obtained at time of inoculation, and at 96 hours, depending on the available antigen specific memory responses and possibly also neutrophil count growth ratios vary among individuals. For instance, HIV infected patients have high growth ratios due to the deficiency of CD four T-cell population, one of the key mediators of cellular immune response to mycobacteria. Importantly, growth ratios are reproducible measures over a period of time In this control experiment, a single donor's blood repeatedly for 12 months.
Here results depict growth ratios calculated from 64 donors bled twice over a period of 12 months. This functional assay paved the way for researchers in the field of tuberculosis to explore the relative contribution of host immune cells to containment of mycobacteria, and it also evaluates the immunogenicity of antigens that might form part of anti TB vaccines in children with and without HIV infection. After watching this video, you should have a good understanding of how to prepare a suitable stock of mycobacteria and obtain a consistent inoculum.
You should be able to prepare blood samples for measurements of bioluminescence in order to calculate the metabolic activity. This reflects the effect of the host immune cells on the microbacteria.
