Finger-stick Blood Sampling Methodology for the Determination of Exercise-induced Lymphocyte Apoptosis

Podsumowanie

Exercise is capable of inducing apoptosis in immune cells. There are various measurement limitations, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment. Demonstrated is a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis.

Streszczenie

Exercise is a physiological stimulus capable of inducing apoptosis in immune cells. To date, various limitations have been identified with the measurement of this phenomenon, particularly relating to the amount of time required to isolate and treat a blood sample prior to the assessment of cell death. Because of this, it is difficult to determine whether reported increases in immune cell apoptosis can be contributed to the actual effect of exercise on the system, or are a reflection of the time and processing necessary to eventually obtain this measurement. In this article we demonstrate a rapid and minimally invasive procedure for the analysis of exercise-induced lymphocyte apoptosis. Unlike other techniques, whole blood is added to an antibody panel immediately upon obtaining a sample. Following the incubation period, red blood cells are lysed and samples are ready to be analyzed. The use of a finger-stick sampling procedure reduces the volume of blood required, and minimizes the discomfort to subjects.

Protokół

1. Finger-Stick Blood Sampling

1. 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).
2. Using Universal Precautions, first sterilize the fingertip with 70% isopropyl alcohol.
3. 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.
4. 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

1. 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.
2. Incubate at room temperature in the dark for 30 minutes.
3. Centrifuge at 1075 x g for 5-10 minutes.
4. Decant, add 300 μL Red Blood Cell Lysis Buffer, and thoroughly vortex.
5. Incubate at room temperature for 15 minutes.
6. Add 300 μL PBS to stop RBC lysis reaction.
7. Centrifuge at 1075 x g for 5-10 minutes.
8. Decant, rack, and add 50 μL binding buffer.
9. 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.

Dyskusje

A minimally invasive sample collection procedure and subsequent analysis is demonstrated for the analysis of both early and late phases of exercise-induced lymphocyte apoptosis. It is also possible to clearly exclude those cells, which are necrotic, and not apoptotic. This procedure overcomes the drawbacks and discomfort associated with invasive venipuncture from the antecubital region at multiple time points prior to, during, and immediately after exercise, or securing a catheter for the duration of the exercise bout. I...

Materiały

Name	Company	Catalog Number	Comments
Flow Cytometry Binding Buffer		eBioscience	00-4222-26
RBC Lysis Buffer		eBioscience	00-4333-57
Lancet Device	Accu Check Soft Touch	Roche Group
Capillary tubes	Heparinized Capillary Tubes	Chase Scientific Glass, Inc.	501
Centrifuge	GmC Lab	Gilson, Inc.	PMS-880
Annexin V-FITC Conjugated		Biovision Inc.	K201-400-1
CD4-PE Conjugated	Anti-human, clone RPA-T4	eBioscience	12-0049-42
CD8-PE Conjugated	Anti-human, clone OKT8	eBioscience	12-0086-73
CD19-PE Conjugated	Anti-human, clone HIB19	eBioscience	12-0199-42
7-AAD Viability Staining Solution		eBioscience	00-6993-50
CD45RA-APC Conjugated	Anti-human, clone HI100	eBioscience	17-0458-42
Flow cytometer	C6	Accuri