Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

In the following protocol, the measurement of the bioenergetic profiles and oxidative burst responses of human blood monocytes, lymphocytes, neutrophils, and platelets will be demonstrated. This is achieved by first collecting the buffy coat and platelet rich plasma fractions from freshly drawn blood. As a second step, the heterogeneous white blood cell population is separated into mononuclear and polymorphonuclear cell layers by density gradient.

Next, the pure monocyte neutrophil and lymphocyte populations are further isolated by max separation and then plated for extracellular flux or XF analysis. Ultimately, the sensitive and reproducible measurement of the mitochondrial function in monocytes, lymphocytes and platelets, as well as the oxidative burst of monocyte and neutrophil oxygen consumption rates can be measured by XF technology. The advantage of the technique that we're going to show you today is that you can prepare from a small blood sample all of the main cells which are in the patient's blood.

These are platelets and the leukocytes in a pure inactivated form and measure their mitochondrial function. This, we think, then can reflect various diseases and states within the patient in a real time. This method could help answer key questions concerning metabolism, such as what is the role of lifestyle, diet, and pathological stress on mitochondrial function.

This protocol combines multiple technologies for the isolation and determination of cellular bioenergetics in various leukocyte populations and in platelets. This step of the protocol is very critical because the density gradient and magnetic separation requires precise pipetting. In addition, you need to be aware of where the cell populations are during the fractionation.

We will demonstrate the isolation of peripheral blood cells and the analysis of cellular bioenergetics on the 24 well extracellular flux analyzer or the XF 24. However, this method can also be performed on the XF 96, At least two hours before beginning the procedure. Hydrate the XF sensor cartridge probes with XF cran solution.

Then spin down the freshly drawn whole blood for 15 minutes at 500 times G and room temperature in a centrifuge with a swinging bucket rotor. Use a transfer pipette to remove the top layer containing the platelet rich plasma until one centimeter of plasma remains above the erythrocyte buffy coat layer and set the plasma aside at room temperature for later processing. Then transfer the Buffy coat to a sterile 50 milliliter conical tube and dilute the white blood cells to 24 milliliters.

With basal RPMI next, add three milliliters of low density, his opaque 1.077 to each of three 15 milliliter conical tubes. Then place the tip of a five milliliter narrow pipette into the bottom of each tube. In turn and slowly release three milliliters of high density, his opaque 1.119.

Now, use an automated pipette on the low power setting to carefully add eight milliliters of the diluted blood to each gradient without disturbing the layers. Blue dye has been added to the low density gradient for visualization of all gradient layers. After separating the cells by centrifugation separately, collect the mononuclear and polymorphonuclear cells with sterile glass pipettes without disturbing the other cell bands.

Pool the mononuclear and polymorphonuclear cell populations from each tube into single sterile 50 milliliter conical tubes for each cell type, and add four volumes of RPMI to both tubes. After spinning down the cells transfer pellets to a 1.5 milliliter tube re pellet and resuspend in 80 microliters of fresh RPMI supplemented with BSA. Then add 20 microliters of magnetic bead labeled anti CD 14 and anti CD 15 antibodies to the mononuclear and polymorphonuclear cell fractions respectively.

Incubate each cell suspension for 15 minutes at four degrees Celsius, and then wash each tube with one milliliter of R-P-M-I-B-S-A media resuspend the pellets in 500 microliters of R-P-M-I-B-S-A. Then apply the cell suspensions to individual R-P-M-I-B-S-A washed LS columns and wash each column three times with three milliliters of R-P-M-I-B-S-A media Collect the cell suspension flow throughs and column washes into sterile tubes to isolate the monocytes and neutrophils, remove the columns from the magnetic field after the final wash and elute the positively selected cells into a sterile tube with five milliliters of R-P-M-I-B-S-A. To isolate the lymphocytes, pellet the flow through wash fraction of the cell column, resuspend the cell pellet in 80 microliters of R-P-M-I-B-S-A, and incubate the cells in 20 microliters of CD 61 and CD 2 35 A antibodies for 15 minutes at four degrees Celsius.

Then repeat the max separation as before, and collect the flow through containing the lymphocytes to isolate the platelets, centrifuge, the reserved platelet rich plasma, and then remove the plasma and wash the cell pellet once with five milliliters of sterile PBS supplemented with one microgram per milliliter of prostaglandin I two resuspend the platelet pellet in one milliliter of fresh PBS prostaglandin I two buffer to plate the isolated monocytes, lymphocytes, neutrophils, and platelets. First count the cells and bring them to an appropriate volume in XF assay medium to allow a seeding density of 2.5 times 10 to the fifth cells per well in a 200 microliter volume. In a 24 well XF cell culture microplate coated with cell T for platelets seed 2.5 times 10 to the seven cells per well in a 200 microliter volume.

After spinning the cells onto the plate, bring the final well volumes up to 660 microliters with XF media and incubate the plates at 37 degrees Celsius for 30 minutes prior to the XF assay during the incubation load, 75 microliters of the assay reagents in the listed order into the XF sensor cartridge injection ports. If oxidative burst measurements are to be obtained, PMA can be injected after the antimycin, a following injection port loading place the cartridge on the extracellular flux analyzer to begin the assay. The bioenergetic profiles of leukocytes and platelets can be determined using the seahorse XF extracellular flux analyzer, which measures real time oxygen consumption in cells non-invasively.

Each cell type is plated on the XF 24 microplate with five replicates as just demonstrated in the table. Representative basal and oxidative burst values as measured during the assay and the average protein concentrations per well are demonstrated by cell type Oxygen consumption rate values are normalized to the total protein content in the corresponding wells and expressed as p moles per minute per microgram of protein. The basal oxygen consumption rate is then established by the first three measurements.

Oligo mycin, an inhibitor of mitochondrial A TP synthase is injected into the XF medium to estimate the oxygen consumption rate coupled to a TP synthesis and is represented as a TP linked the residual oxygen consumption rate minus the non mitochondrial oxygen consumption rate can be attributed to proton leak. Addition of the uncoupling FCCP allows determination of the maximal oxygen consumption rate, followed by injection of antimycin a and an inhibitor of mitochondrial respiration to measure the non mitochondrial sources of oxygen consumption. Reserve capacity is a measure of the amount of additional work that the mitochondria can perform under increased energy demand and can be calculated as the difference between the maximum rate of respiration and the basal in order to determine the oxidative burst capacity of monocytes and neutrophils.

P-M-A-A-P-K-C agonist is injected and the increase in oxygen consumption rate is linked to oxidant production by N-A-D-P-H oxidase Once mastered, this technique can be done in as little as three hours following blood collection, not including the assay itself if it is performed properly. While attempting this procedure, it is important to maintain sterile conditions by performing each step in a biological safety cabinet and using sterile room temperature buffers and medias. Following this procedure, we can do other protocols, such as fax analysis to determine the purity of the cells, the activation state of leukocytes, and also to determine the cellular bioenergetics in leukocytes subpopulations.

After watching this video, you should now have a fairly clear idea about how to prepare platelets, lymphocytes, monocytes, and neutrophils, and put them into the seahorse and measure the oxidative burst and mitochondrial function from these data. Then you can analyze your patient populations and determine how their bioenergetics are changing in real time. Don't forget that working with human patient samples can be extremely hazardous.

Therefore, you need to wear personal protective equipment as well as use a biological safety cabinet during this procedure.