Combined boyden-flow cytometry assay improves quantification and provides phenotypification of leukocyte chemotaxis

Abstract

Chemotaxis has been studied by classical methods that measure chemotactic and random motility responses in vitro, but these methods do not evaluate the total number and phenotype of migrating leukocytes simultaneously. Our objective was to develop and validate a novel assay, combined Boyden-flow cytometry chemotaxis assay (CBFCA), for simultaneous quantification and phenotypification of migrating leukocytes. CBFCA exhibited several important advantages in comparison to the classic Boyden chemotaxis assay (CBCA): 1) improved precision (intra-assay coefficients of variation (CVs): CBFCA-4.7 and 4.8% vs. CBCA-30.1 and 17.3%; inter-observer CVs: CBFCA-3.6% vs. CBCA 30.1%); 2) increased recovery of cells, which increased assay to provide increased sensitivity; 3) high specificity for determining the phenotype of migrating/attracted leukocytes; and 4) reduced performance time (CBFCA 120 min vs. CBCA 265 min). Other advantages of CBFCA are: 5) robustness, 6) linearity, 7) eliminated requirement for albumin and, importantly, 8) enabled recovery of migrating leukocytes for subsequent studies. This latter feature is of great benefit in the study of migrating leukocyte subsets. We conclude that the CBFCA is a novel and improved technique for experiments focused on understanding leukocyte trafficking during the inflammatory response.

Figure 1. CBCA & CBFCA.

A) CBCA includes a permeable polycarbonate membrane and a second cell impermeable cellulose membrane directly beneath to trap attracted cells. These membranes separate the upper well containing leukocytes and the lower well containing the chemotactic. Attracted leukocytes are counted in the cellulose membrane under light microscopy in different focal planes (magnification). White arrows represent the leukocytes trapped and agglutinated into the cellulose membrane, and the yellow arrow represents its pores. Light microscopy: magnification x400; Red bar 10 µm. B) CBFCA includes a single polycarbonate membrane between the wells. Attracted leukocytes in the lower well are counted by flow cytometry and their number increases lineally with the length of interval. The leukocyte phenotype can also be determined in this step.

Figure 2. Precision: Intra-assay and inter-observer variation.

A&B) Two sets of assays were performed by CBCA or CBFCA, set 1 includes 12 assays (gray diamonds) and set 2 includes 15 assays (black squares). Intra-assay coefficient of variation (CV) in CBCA was higher than in CBFCA in both sets (30.1 and 17.3% vs 4.7 and 4.8%). D&C) Three of these assays were counted by three different observers/operators using microscopy or flow cytometry. The inter-observer CV in CBCA was higher than in CBFCA (30.1 vs 3.6%). Data are shown as means±SD of three independent assays counted in triplicate.

Figure 3. Specificity of CBFCA.

CBFCA involves the determination of the leukocyte phenotype using conjugated specific antibodies and flow cytometry. A) Representative dot-plots showing the dual parameters used for the flow cytometric analysis: CD45+ for total leukocytes, CD45+CD3+ for T-lymphocytes, CD45+CD14+ for monocytes, CD45+CD56+ for NK cells and CD45+CD19+ for B lymphocytes. B) Specific leukocyte chemotactic activity of 10⁻⁶ M fMLP and plasma. Leukocyte subset proportions were significantly different before and after the assay. Data shown are means±SD of five independent assays in triplicate. Significance was determined by ANOVA and Games-Howell test.

Figure 4. A) Robustness of CBFCA.

To investigate the robustness of CBFCA the leukocyte chemotactic activity of undiluted and diluted plasma was determined in time course experiments. CBFCA was able to determine the biological chemotactic activity of diluted and undiluted plasma at 60, 90 and 120 min. B) Robustness of the incubation time in CBFCA. CBFCAs were performed at different incubation times (60, 90 and 120 min) using total leukocytes and plasma. All the leukocyte subsets reached the top of their migration within 90 min and their proportions did not change at 120 min of incubation time. Granulocytes and B-lymphocytes completed their migration within the first 60 min. Data shown are means±SD of five independent assays in triplicate. Means with same symbol are significantly different. Significance was determined by ANOVA and Games-Howell tests (p≤†‡γ¥ 0.0001, λ 0.005).

Figure 5. Linearity of CBFCA.

CBFCAs were performed using 10⁻³ and 10⁻⁶ M fMLP as chemotactic and purified neutrophils (A) and monocytes (B) at different concentrations. The number of attracted neutrophils and monocytes increased lineally with the starting number of cells. Significantly more leukocytes were attracted by 10⁻³ M fMLP than by 10⁻⁶ M fMLP in all the cases. Data shown are means±SD of four independent assays in triplicate. Significance was determined by T test.

Figure 6. Effect of HA on leukocyte chemotaxis.

CBFCAs were performed using medium or 10⁻⁶ M fMLP, in the absence or presence of 0.1% and 2% HA. The addition of 2% HA to the medium and 10⁻⁶ M fMLP attracted more leukocytes; however, it also masked the chemotactic activity of 10⁻⁶ M fMLP. Data are shown as means±SD of five independent assays in triplicate. Means with same symbol are significantly different. Significance was determined by ANOVA, Games-Howell tests and T test (p = †‡ 0.027).