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A Simple and Efficient Method for Preparing Cell Slides and Staining without Using Cytocentrifuge and Cytoclips.

Hu X, Laguerre V, Packert D, Nakasone A, Moscinski L - Int J Cell Biol (2015)

Bottom Line: This method is based on the principle that a regular cell centrifuge can be used to concentrate cells harvested from cell culture and then deposit the concentrated cell suspension to a slide evenly by using a cell spreader, followed by cell staining.The method presented is simple, rapid, economic, and efficient.This method may also avoid a possible change in cell morphology induced by cytocentrifuge.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of Arts & Sciences, Barry University, Miami Shores, FL, USA.

ABSTRACT
Cell staining is a necessary and useful technique for visualizing cell morphology and structure under a microscope. This technique has been used in many areas such as cytology, hematology, oncology, histology, virology, serology, microbiology, cell biology, and immunochemistry. One of the key pieces of equipment for preparing a slide for cell staining is cytology centrifuge (cytocentrifuge) such as cytospin. However, many small labs do not have this expensive equipment and its accessory, cytoclips (also expensive relatively), which makes them difficult to study cell cytology. Here we present an alternative method for preparing a slide and cell staining in the absence of a cytocentrifuge (and cytoclips). This method is based on the principle that a regular cell centrifuge can be used to concentrate cells harvested from cell culture and then deposit the concentrated cell suspension to a slide evenly by using a cell spreader, followed by cell staining. The method presented is simple, rapid, economic, and efficient. This method may also avoid a possible change in cell morphology induced by cytocentrifuge.

No MeSH data available.


Related in: MedlinePlus

TF-1a cells stained with DIPP Quick Stains. TF-1a cells were treated with PMA for 48 hours, after which the cells were collected and slides were prepared following the method described in Section 2. The staining method followed manufacture's instruction (×1000).
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fig3: TF-1a cells stained with DIPP Quick Stains. TF-1a cells were treated with PMA for 48 hours, after which the cells were collected and slides were prepared following the method described in Section 2. The staining method followed manufacture's instruction (×1000).

Mentions: In a basic research, the cell concentrations in culture are usually under 106/mL in order to prevent contact inhibition; in particular, the cells need to be treated with growth factors, interleukins, and stimuli for few hours to days. Therefore, these cells need to be concentrated before applying them to a slide. In the absence of a cytology centrifuge, we used regular cell centrifuge to concentrate cells harvested from the culture (see Materials and Methods). Subsequently, the 1-2 drops of the concentrated cells were dropped to the central area of the slide with a plastic transfer pipette, after which a glass spreader was used to move cells around to form a thin and even layer. The glass spreader was made from a glass transfer pipette over an alcohol burner for a few seconds to 1 min. Figure 1 shows an image of the glass spreader during the process of applying cells to a slide. In order to test whether the slides made by using this noncytocentrifuge method and the glass spreader are good enough for cytology study, three types of human myeloid leukemia cells were applied for preparation of slides and two types of stain methods were used for staining. The first cell line is TF-1a. TF-1a is a factor-independent cell line derived from the human factor-dependent erythroid leukemia TF-1 cell line but appears less mature than TF-1, because TF-1a is CD34 positive (+) and CD38 is negative (−), whereas TF-1 is CD34 + and CD38 +. TF-1a cells have big nucleus/cytoplasm ratio in the absence of differentiation. Our previous experiments have demonstrated that TF-1a cells are able to respond to PMA and can be induced to macrophage-like differentiation [6]. This cell line is a good model to study cell differentiation. As shown in Figure 2, cells treated with Giemsa (a) show good contrast. The dye stains cytoplasm, membrane, and nuclei. After addition of PMA to the cells for 72 h, the ratio of nucleus/cytoplasm is significantly reduced. The cells in (b) are stained with methylene blue and eosin. The methylene blue stain makes nuclei more visible although it stains both nuclei and cytoplasm; the eosin mainly colors the cytoplasm and cell membranes. From Figure 2, 90% of the control cells (without PMA) show a blue color, indicating the majority of the cells are nucleus, which gives a larger ratio of nucleus/cytoplasm. After treatment of the cells with PMA for 72 hours, the pink color from the eosin stain becomes dominant indicating the major part of the cells has changed to cytoplasm due to a decrease in the size of nucleus, leading to a smaller ratio of nucleus/cytoplasm. Next, we tested whether the slides prepared by this method gave equivalent effect to a commercial stain kit; the DIPP Quick Stain was used. This kit contains two dyes: methylene and eosin. As indicated in Figure 3, the DIPP KWIK smears show similar images and color changes to those slides stained with the reagents shown in Figure 2. Overall, the smear images from the slides prepared by our noncytocentrifuge method are equivalent to the slides prepared from cytocentrifuge in our previous experiments [6]. The second cell line tested is TF-1. As described above, TF-1 is more mature than TF-1a. This cell line is insensitive to PMA-induced differentiation but can be induced to apoptosis in response to Bay 11-7085, a NF-κB inhibitor that has been reported to induce cell death in several cell types [7, 8]. Figure 4(a) shows the morphology of TF-1 cells in the absence or presence of Bay 11-7085 for 48 hours. The control cells (without Bay 11-7085) show normal cell morphology with complete cell membrane. The treatment of the cells with Bay 11-7085 caused significant changes in the cell morphology. At a concentration of 10 μM, Bay 11-7085 caused the cells shrinkage and blebbing. About 50% of cells were completely broken down when the concentration was increased to 20 μM (Figure 3). The third cell line tested is MV4-11 cells. MV4-11 is CD38+/CD34+ and is the most matured cell line as compared with TF-1a and TF-1. This cell line does not respond to PMA-induced differentiation and Bay 11-7085 induced apoptosis. There are no significant changes in the morphology between the control cells (without Bay 11-7085) and the cells treated with Bay 11-7085 for 48 hours (Figure 4(b)).


A Simple and Efficient Method for Preparing Cell Slides and Staining without Using Cytocentrifuge and Cytoclips.

Hu X, Laguerre V, Packert D, Nakasone A, Moscinski L - Int J Cell Biol (2015)

TF-1a cells stained with DIPP Quick Stains. TF-1a cells were treated with PMA for 48 hours, after which the cells were collected and slides were prepared following the method described in Section 2. The staining method followed manufacture's instruction (×1000).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4664808&req=5

fig3: TF-1a cells stained with DIPP Quick Stains. TF-1a cells were treated with PMA for 48 hours, after which the cells were collected and slides were prepared following the method described in Section 2. The staining method followed manufacture's instruction (×1000).
Mentions: In a basic research, the cell concentrations in culture are usually under 106/mL in order to prevent contact inhibition; in particular, the cells need to be treated with growth factors, interleukins, and stimuli for few hours to days. Therefore, these cells need to be concentrated before applying them to a slide. In the absence of a cytology centrifuge, we used regular cell centrifuge to concentrate cells harvested from the culture (see Materials and Methods). Subsequently, the 1-2 drops of the concentrated cells were dropped to the central area of the slide with a plastic transfer pipette, after which a glass spreader was used to move cells around to form a thin and even layer. The glass spreader was made from a glass transfer pipette over an alcohol burner for a few seconds to 1 min. Figure 1 shows an image of the glass spreader during the process of applying cells to a slide. In order to test whether the slides made by using this noncytocentrifuge method and the glass spreader are good enough for cytology study, three types of human myeloid leukemia cells were applied for preparation of slides and two types of stain methods were used for staining. The first cell line is TF-1a. TF-1a is a factor-independent cell line derived from the human factor-dependent erythroid leukemia TF-1 cell line but appears less mature than TF-1, because TF-1a is CD34 positive (+) and CD38 is negative (−), whereas TF-1 is CD34 + and CD38 +. TF-1a cells have big nucleus/cytoplasm ratio in the absence of differentiation. Our previous experiments have demonstrated that TF-1a cells are able to respond to PMA and can be induced to macrophage-like differentiation [6]. This cell line is a good model to study cell differentiation. As shown in Figure 2, cells treated with Giemsa (a) show good contrast. The dye stains cytoplasm, membrane, and nuclei. After addition of PMA to the cells for 72 h, the ratio of nucleus/cytoplasm is significantly reduced. The cells in (b) are stained with methylene blue and eosin. The methylene blue stain makes nuclei more visible although it stains both nuclei and cytoplasm; the eosin mainly colors the cytoplasm and cell membranes. From Figure 2, 90% of the control cells (without PMA) show a blue color, indicating the majority of the cells are nucleus, which gives a larger ratio of nucleus/cytoplasm. After treatment of the cells with PMA for 72 hours, the pink color from the eosin stain becomes dominant indicating the major part of the cells has changed to cytoplasm due to a decrease in the size of nucleus, leading to a smaller ratio of nucleus/cytoplasm. Next, we tested whether the slides prepared by this method gave equivalent effect to a commercial stain kit; the DIPP Quick Stain was used. This kit contains two dyes: methylene and eosin. As indicated in Figure 3, the DIPP KWIK smears show similar images and color changes to those slides stained with the reagents shown in Figure 2. Overall, the smear images from the slides prepared by our noncytocentrifuge method are equivalent to the slides prepared from cytocentrifuge in our previous experiments [6]. The second cell line tested is TF-1. As described above, TF-1 is more mature than TF-1a. This cell line is insensitive to PMA-induced differentiation but can be induced to apoptosis in response to Bay 11-7085, a NF-κB inhibitor that has been reported to induce cell death in several cell types [7, 8]. Figure 4(a) shows the morphology of TF-1 cells in the absence or presence of Bay 11-7085 for 48 hours. The control cells (without Bay 11-7085) show normal cell morphology with complete cell membrane. The treatment of the cells with Bay 11-7085 caused significant changes in the cell morphology. At a concentration of 10 μM, Bay 11-7085 caused the cells shrinkage and blebbing. About 50% of cells were completely broken down when the concentration was increased to 20 μM (Figure 3). The third cell line tested is MV4-11 cells. MV4-11 is CD38+/CD34+ and is the most matured cell line as compared with TF-1a and TF-1. This cell line does not respond to PMA-induced differentiation and Bay 11-7085 induced apoptosis. There are no significant changes in the morphology between the control cells (without Bay 11-7085) and the cells treated with Bay 11-7085 for 48 hours (Figure 4(b)).

Bottom Line: This method is based on the principle that a regular cell centrifuge can be used to concentrate cells harvested from cell culture and then deposit the concentrated cell suspension to a slide evenly by using a cell spreader, followed by cell staining.The method presented is simple, rapid, economic, and efficient.This method may also avoid a possible change in cell morphology induced by cytocentrifuge.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, College of Arts & Sciences, Barry University, Miami Shores, FL, USA.

ABSTRACT
Cell staining is a necessary and useful technique for visualizing cell morphology and structure under a microscope. This technique has been used in many areas such as cytology, hematology, oncology, histology, virology, serology, microbiology, cell biology, and immunochemistry. One of the key pieces of equipment for preparing a slide for cell staining is cytology centrifuge (cytocentrifuge) such as cytospin. However, many small labs do not have this expensive equipment and its accessory, cytoclips (also expensive relatively), which makes them difficult to study cell cytology. Here we present an alternative method for preparing a slide and cell staining in the absence of a cytocentrifuge (and cytoclips). This method is based on the principle that a regular cell centrifuge can be used to concentrate cells harvested from cell culture and then deposit the concentrated cell suspension to a slide evenly by using a cell spreader, followed by cell staining. The method presented is simple, rapid, economic, and efficient. This method may also avoid a possible change in cell morphology induced by cytocentrifuge.

No MeSH data available.


Related in: MedlinePlus