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Direct observation of CD4 T cell morphologies and their cross-sectional traction force derivation on quartz nanopillar substrates using focused ion beam technique.

Kim DJ, Kim GS, Hyung JH, Lee WY, Hong CH, Lee SK - Nanoscale Res Lett (2013)

Bottom Line: Direct observations of the primary mouse CD4 T cell morphologies, e.g., cell adhesion and cell spreading by culturing CD4 T cells in a short period of incubation (e.g., 20 min) on streptavidin-functionalized quartz nanopillar arrays (QNPA) using a high-content scanning electron microscopy method were reported.Furthermore, we first demonstrated cross-sectional cell traction force distribution of surface-bound CD4 T cells on QNPA substrates by culturing the cells on top of the QNPA and further analysis in deflection of underlying QNPA via focused ion beam-assisted technique.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physics, Chung-Ang University, Seoul 156-756, Republic of Korea. sangkwonlee@cau.ac.kr.

ABSTRACT
Direct observations of the primary mouse CD4 T cell morphologies, e.g., cell adhesion and cell spreading by culturing CD4 T cells in a short period of incubation (e.g., 20 min) on streptavidin-functionalized quartz nanopillar arrays (QNPA) using a high-content scanning electron microscopy method were reported. Furthermore, we first demonstrated cross-sectional cell traction force distribution of surface-bound CD4 T cells on QNPA substrates by culturing the cells on top of the QNPA and further analysis in deflection of underlying QNPA via focused ion beam-assisted technique.

No MeSH data available.


Related in: MedlinePlus

Schematic diagram of QNPA fabrication and separation processes. (a) Schematic diagram outlining the fabrication of quartz nanopillar arrays (QNPAs) where two different sizes of PS were presented for specific example. (b) Surface functionalization including APTES, GA, and STR reactions of QNPAs on a quartz substrate. (c) Schematic diagram of specific CD4 T cell separation process from introduced cell suspension containing CD4 T, CD8 T, NK, and NKT cells from primary mouse splenocytes.
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Figure 1: Schematic diagram of QNPA fabrication and separation processes. (a) Schematic diagram outlining the fabrication of quartz nanopillar arrays (QNPAs) where two different sizes of PS were presented for specific example. (b) Surface functionalization including APTES, GA, and STR reactions of QNPAs on a quartz substrate. (c) Schematic diagram of specific CD4 T cell separation process from introduced cell suspension containing CD4 T, CD8 T, NK, and NKT cells from primary mouse splenocytes.

Mentions: Figure 1a,b shows a schematic illustration of QNPA fabrication processes and further surface functionalization processes, respectively. First, the fabrication process went through a series of process including polystyrene (PS) monolayer deposition, PS size reduction, Ni metal deposition, PS lift-off, additional Cr metal deposition, Ni lift-off, and final reactive ion etching process we have improved previously [19,20]. In addition, the surface of QNPA substrates treated by O2 plasma was then applied by three-step surface functionalization processes using 1% (v/v) (3-aminopropyl)-triethoxysilane (APTES) in ethanol for 30 min at room temperature, 12.5% (v/v) glutaraldehyde (GA) in distilled water for 4 h on a 2D rocker, and approximately 50-μg/mL STR solution in phosphate buffered saline (PBS) overnight in an incubator (37°C, 5% CO2). We used this surface-functionalized method on nanotopographic substrates to separate targeting specific cells (e.g., CD4 T cells) among different kinds of cells via the novel STR-biotin conjugation technique to capture the incoming targeting cells in PBS solution as we have developed previously [20,21]. The T lymphocytes were mouse CD4 T cells from whole mouse splenocytes. Mouse splenocytes (approximately 105 cells per sample) containing CD4 T, CD8 T, natural killer (NK), and natural killer T (NKT) cells were prepared from the spleen of C57BL/6/mice (Nara Biotech, Seoul, South Korea) [22]. Prior to introducing the cell suspension in PBS solution onto the QNPA substrates (0.7 cm × 0.7 cm), the cell population (Figure 1c) with a final volume of approximately 30 μl was first reacted with biotin anti-mouse CD4 antibody and incubated at 4°C for 20 min. The cell suspension containing T cells and other cells pre-reacted with biotin anti-mouse CD4 antibody was then introduced on the STR-functionalized QNPA substrates. Following 20 min of incubation at 4°C in a refrigerator, where the CD4 T cells were in a very early stage of cell adhesion on the QNPA substrates, unbound cells were removed by rinsing with PBS solution. This step was repeated at least five times for 10 min on a 2D rocker to completely remove nonspecifically unbound cells from the QNPA substrates (third image in Figure 1c). Our experiments were focused on targeted CD4 T cell adhesion on STR-functionalized QNPA substrates at a very early stage of cell adhesion (<20 min). To examine the morphologies of the captured CD4 T cells bound on STR-conjugated QNPA substrates, SEM observation was performed. For the SEM observation of the captured cells on QNPA substrate, a series of cell-fixing processes are required as follows. The T cells were first fixed with 4% GA in the refrigerator for 2 h, followed by a post-fix process using 1% osmium tetroxide for 2 h. The T cells were then dehydrated through a series of ethanol concentrations (25%, 50%, 75%, 95%, and 100%) and slowly dried at vacuum-connected desiccators for 24 h [21,23,24]. According to a previous report, the average conventional fixed material, after all steps of preservation, retained 72% of its initial size [25]. Once the samples were dry in the desiccators, the surface-bound T cells were sputter-coated with platinum before the SEM measurement was performed.


Direct observation of CD4 T cell morphologies and their cross-sectional traction force derivation on quartz nanopillar substrates using focused ion beam technique.

Kim DJ, Kim GS, Hyung JH, Lee WY, Hong CH, Lee SK - Nanoscale Res Lett (2013)

Schematic diagram of QNPA fabrication and separation processes. (a) Schematic diagram outlining the fabrication of quartz nanopillar arrays (QNPAs) where two different sizes of PS were presented for specific example. (b) Surface functionalization including APTES, GA, and STR reactions of QNPAs on a quartz substrate. (c) Schematic diagram of specific CD4 T cell separation process from introduced cell suspension containing CD4 T, CD8 T, NK, and NKT cells from primary mouse splenocytes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750221&req=5

Figure 1: Schematic diagram of QNPA fabrication and separation processes. (a) Schematic diagram outlining the fabrication of quartz nanopillar arrays (QNPAs) where two different sizes of PS were presented for specific example. (b) Surface functionalization including APTES, GA, and STR reactions of QNPAs on a quartz substrate. (c) Schematic diagram of specific CD4 T cell separation process from introduced cell suspension containing CD4 T, CD8 T, NK, and NKT cells from primary mouse splenocytes.
Mentions: Figure 1a,b shows a schematic illustration of QNPA fabrication processes and further surface functionalization processes, respectively. First, the fabrication process went through a series of process including polystyrene (PS) monolayer deposition, PS size reduction, Ni metal deposition, PS lift-off, additional Cr metal deposition, Ni lift-off, and final reactive ion etching process we have improved previously [19,20]. In addition, the surface of QNPA substrates treated by O2 plasma was then applied by three-step surface functionalization processes using 1% (v/v) (3-aminopropyl)-triethoxysilane (APTES) in ethanol for 30 min at room temperature, 12.5% (v/v) glutaraldehyde (GA) in distilled water for 4 h on a 2D rocker, and approximately 50-μg/mL STR solution in phosphate buffered saline (PBS) overnight in an incubator (37°C, 5% CO2). We used this surface-functionalized method on nanotopographic substrates to separate targeting specific cells (e.g., CD4 T cells) among different kinds of cells via the novel STR-biotin conjugation technique to capture the incoming targeting cells in PBS solution as we have developed previously [20,21]. The T lymphocytes were mouse CD4 T cells from whole mouse splenocytes. Mouse splenocytes (approximately 105 cells per sample) containing CD4 T, CD8 T, natural killer (NK), and natural killer T (NKT) cells were prepared from the spleen of C57BL/6/mice (Nara Biotech, Seoul, South Korea) [22]. Prior to introducing the cell suspension in PBS solution onto the QNPA substrates (0.7 cm × 0.7 cm), the cell population (Figure 1c) with a final volume of approximately 30 μl was first reacted with biotin anti-mouse CD4 antibody and incubated at 4°C for 20 min. The cell suspension containing T cells and other cells pre-reacted with biotin anti-mouse CD4 antibody was then introduced on the STR-functionalized QNPA substrates. Following 20 min of incubation at 4°C in a refrigerator, where the CD4 T cells were in a very early stage of cell adhesion on the QNPA substrates, unbound cells were removed by rinsing with PBS solution. This step was repeated at least five times for 10 min on a 2D rocker to completely remove nonspecifically unbound cells from the QNPA substrates (third image in Figure 1c). Our experiments were focused on targeted CD4 T cell adhesion on STR-functionalized QNPA substrates at a very early stage of cell adhesion (<20 min). To examine the morphologies of the captured CD4 T cells bound on STR-conjugated QNPA substrates, SEM observation was performed. For the SEM observation of the captured cells on QNPA substrate, a series of cell-fixing processes are required as follows. The T cells were first fixed with 4% GA in the refrigerator for 2 h, followed by a post-fix process using 1% osmium tetroxide for 2 h. The T cells were then dehydrated through a series of ethanol concentrations (25%, 50%, 75%, 95%, and 100%) and slowly dried at vacuum-connected desiccators for 24 h [21,23,24]. According to a previous report, the average conventional fixed material, after all steps of preservation, retained 72% of its initial size [25]. Once the samples were dry in the desiccators, the surface-bound T cells were sputter-coated with platinum before the SEM measurement was performed.

Bottom Line: Direct observations of the primary mouse CD4 T cell morphologies, e.g., cell adhesion and cell spreading by culturing CD4 T cells in a short period of incubation (e.g., 20 min) on streptavidin-functionalized quartz nanopillar arrays (QNPA) using a high-content scanning electron microscopy method were reported.Furthermore, we first demonstrated cross-sectional cell traction force distribution of surface-bound CD4 T cells on QNPA substrates by culturing the cells on top of the QNPA and further analysis in deflection of underlying QNPA via focused ion beam-assisted technique.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physics, Chung-Ang University, Seoul 156-756, Republic of Korea. sangkwonlee@cau.ac.kr.

ABSTRACT
Direct observations of the primary mouse CD4 T cell morphologies, e.g., cell adhesion and cell spreading by culturing CD4 T cells in a short period of incubation (e.g., 20 min) on streptavidin-functionalized quartz nanopillar arrays (QNPA) using a high-content scanning electron microscopy method were reported. Furthermore, we first demonstrated cross-sectional cell traction force distribution of surface-bound CD4 T cells on QNPA substrates by culturing the cells on top of the QNPA and further analysis in deflection of underlying QNPA via focused ion beam-assisted technique.

No MeSH data available.


Related in: MedlinePlus