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1. Finger-Stick Blood Sampling
- Collection of whole blood is typically taken at rest for a baseline measurement (usually following 10-15 min seated rest), during or immediately following an exercise bout, and throughout the post-exercise period (generally from 1-3 hours following cessation of the exercise).
- Using Universal Precautions, first sterilize the fingertip with 70% isopropyl alcohol.
- Next, puncture the site using an automated lancet or similar device. Although we use a 30-gauge lancet to provide for subject comfort, a larger gauge needle could be utilized.
- Wipe away the first drop of blood, and then collect blood into capillary tubes or microvettes treated with lithium heparin to prevent clotting.
2. Cell Phenotyping and Apoptosis Staining
- Add 10 μL heparinized whole blood to titred antibody panel in 250 μL binding buffer (see table 1 schematic). We have used a 1:20 dilution factor with success, but each laboratory should titre their reagents to determine the best working solution.
Table 1. Antibody panel for the determination of exercise-induced apoptosis in leukocyte subsets. Early apoptosis was defined by the expression of Annexin V, while late apoptosis was defined by double positive labeling with Annexin V and 7-AAD. Necrotic cells were Annexin V- and 7-AAD+ cells only.
| Tube 1 | Tube 2 | Tube 3 | Tube 4 |
| Cells only tube | Annexin V - FITC | Annexin V - FITC | Annexin V - FITC |
| | Anti Human CD4 - PE | Anti Human CD8 - PE | Anti Human CD19 - PE |
| | 7-AAD | 7-AAD | 7-AAD |
| | Anti Human CD45RA - APC | Anti Human CD45RA - APC | |
7-AAD = 7-Amino-actinomycin D, APC = Allophycocyanin, CD4 = Helper T lymphocytes, CD8 = Suppressor/cytotoxic T lymphocytes, CD19 = B lymphocytes, CD45RA = naíve cell subsets, FITC = Fluorescein isothiocyanate, PE = Phycoerythrin.
- Incubate at room temperature in the dark for 30 minutes.
- Centrifuge at 1075 x g for 5-10 minutes.
- Decant, add 300 μL Red Blood Cell Lysis Buffer, and thoroughly vortex.
- Incubate at room temperature for 15 minutes.
- Add 300 μL PBS to stop RBC lysis reaction.
- Centrifuge at 1075 x g for 5-10 minutes.
- Decant, rack, and add 50 μL binding buffer.
- Analyze by flow cytometry.
It is recommended that at a minimum, the following controls be utilized: a cells only tube to serve as a negative control in detecting background autofluorescence, and tubes containing each individual fluorochrome to set compensation during initial set up of the flow cytometer. In addition, we have used compensation standard beads successfully to set up our experiments.