For microscopic analysis, cells were washed in phosphate-buffered saline (PBS) and fixed in PBS containing 3.7% formaldehyde (Merck, Rahway, NJ, USA) for 10 min. Cells were resuspended in 500 µL of PBS, and cytocentrifuged on clean slides (using a Cytospin 4 Cytocentrifuge, Thermo Fisher Scientific, at 900 rpm for 4 min). Slides were then immersed in liquid nitrogen for 5 min, transferred to PBS containing 0.1% TritonX-100 for 30 min, and then transferred to PBS containing 3% BSA (AppliChem, Darmstadt, Germany) for 30 min. The slides were immunostained using the following primary antibodies, all diluted in a 1:1 mixture of PBT and 0.3% BSA: chicken anti-GFP (1:200, Thermo Fisher Scientific, PA1-9533) and mouse anti-Fibrillarin (1:200, Thermo Fisher Scientific, MA1-22000). Primary antibodies were detected via incubation for 1 h with Alexa Fluor 488-conjugated goat anti-chicken IgG (1:300, Thermo Fisher Scientific, A11039) and FITC-conjugated goat anti-mouse IgG (1:40, Sigma-Aldrich, Burlington, MA, USA, F8264). Slides were then mounted in Vectashield antifade mounting medium containing 4.6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Newark, CA, USA). Images of fixed cells were captured using Zeiss Axio Imager M2 equipped with an EC Plan-Neofluar 100×/1.30 oil lens (Carl Zeiss Microscopy, Oberkochen, Germany) and with an AxioCam 506 mono (D) camera.
Cytospin 4 cytocentrifuge
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Australia, Germany, Japan
The Cytospin 4 Cytocentrifuge is a laboratory instrument designed for the preparation of cell samples for microscopic examination. It uses a controlled centrifugal force to deposit a thin, even layer of cells onto a microscope slide, enabling efficient and consistent sample preparation.
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Lab products found in correlation
May gr nwald giemsa, by Merck Group (4 mentions)
Microscope slide, by Thermo Fisher Scientific (4 mentions)
Diff quik stain set, by Siemens (4 mentions)
Trypan blue, by Thermo Fisher Scientific (3 mentions)
Reastain quick diff kit, by Gentaur (3 mentions)
Bx51 microscope, by Olympus (3 mentions)
Bx53 microscope, by Olympus (3 mentions)
Giemsa, by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Cytofunnel, by Thermo Fisher Scientific (3 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Bovine serum albumin (bsa), by AppliChem (2 mentions)
Plan fluor objective, by Nikon (2 mentions)
Eclipse ni microscope, by Nikon (2 mentions)
Nis elements software, by Nikon (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Mgk s mounting solution, by Matsunami (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
Ax10 inverted microscope, by Zeiss (2 mentions)
Diff quik staining set, by Siemens (2 mentions)
105 protocols using cytospin 4 cytocentrifuge
1
Immunostaining and Fluorescence Microscopy
2
Flow Cytometry and Diff-Quik Staining
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Cells were analyzed by flow cytometry as described by Olschok et al. (2021) and Toledo et al. (2021) (link) (Figure S4 B; Table S1 ) and analyzed with FACS Canto II or LSR Fortessa and FlowJo software (all BD Bioscience, Franklin Lakes, NJ). For the analysis of protein biosynthesis with flow cytometry, the protein synthesis assay kit (ab239725, Abcam, Cambridge, UK) was used.
Diff-Quik staining cells were centrifuged onto glass slides in the Cytospin 4 cytocentrifuge (Thermo Fisher Scientific). Cells were fixed with methanol, stained with Diff-Quik (Medion Diagnostics, Düdingen, Switzerland), and mounted with Entellan (Merck, Rahway, NJ).
Diff-Quik staining cells were centrifuged onto glass slides in the Cytospin 4 cytocentrifuge (Thermo Fisher Scientific). Cells were fixed with methanol, stained with Diff-Quik (Medion Diagnostics, Düdingen, Switzerland), and mounted with Entellan (Merck, Rahway, NJ).
3
Quantifying CD34+ Cell Deposition
4
Cytospin Preparation of Mouse Eosinophils and Human Stromal Vascular Fraction
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For mouse cytospin, eosinophils were sorted into 1.5 ml tubes by fluorescence-activated cell sorting from adipose tissue as CD45+SiglecF+Gr1- cells. The sorted cells were centrifuged at 300 g for 5 minutes at 4°C, washed once with 1 ml of cold PBS, centrifuged at 300 g for 5 minutes at 4°C, and resuspended in 100 ml of cold PBS. This 100 ml cell suspension was loaded into a cytofunnel and spun down onto a microscope slide using a Thermo Scientific Cytospin 4 Cytocentrifuge (Thermo #A78300003) at 800 rpm on medium acceleration for 3 minutes at room temperature. Slides were immediately fixed in 100% methanol for 5 minutes and then stored at 4°C until their subsequent staining.
For human cytospin, 100 µl of the whole stromovascular fraction (SVF) suspension was loaded into a cytofunnel. Cytospin and cell fixation was performed as described above.
For human cytospin, 100 µl of the whole stromovascular fraction (SVF) suspension was loaded into a cytofunnel. Cytospin and cell fixation was performed as described above.
5
Quantification of Nanoparticle Uptake in Jurkat Cells
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Jurkat cells 1 × 105 per well in 200 μL (at 5 × 105 cells/mL) were cultured without nanoparticles or in the presence of nanoparticles at 1.5 μg/mL or 15 μg/mL, subsequently washed with phosphate-buffered saline (PBS), and fixed in a solution containing 4% formaldehyde in PBS at room temperature, for 30 min. After an additional washing step, cells were centrifuged at 900 rpm for 5 min on adhesion poly-lysine slides, Polysine® VWR Collection by means of a Cytospin™ 4 Cytocentrifuge (ThermoFisher Scientific), washed and mounted with Fluoro-Gel mounting medium (Catalog #17985-10, Electron Microscopy Sciences). Slides were analyzed on an Olympus Confocal microscope. Cells were excited at λ405, and emission was collected at λ413–510 with a × 40 magnification. Images were analyzed with the Fiji distribution software (Schindelin et al. 2012 (link)) of ImageJ (Schneider et al. 2012 (link)). Percentage of blue (413–510λ)-emitting cells from total cell (from a transmission micrograph) were obtained from three independent experiments and analyzed by one-way ANOVA using the XLSTAT® Excell® plug in.
6
Cytospin and Immunofluorescence for 2D and 3D T Cell Cultures
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A Cytospin 4 Cytocentrifuge (Thermo Fisher scientific) was employed to obtain a CD4+ and CD8+ T cells on the glass slides for the 2D control group since they grow in suspension. The obtained samples were fixed in 4% Paraformaldehyde (PFA) for 15 min and permeabilized in 0.3% TRITON X-100 (Sigma-Aldrich) for 10 min at RT. Then, the samples were incubated in 5% Bovine Serum Albumin (BSA) blocking solution for 30 min to saturate the nonspecific sites, and incubated with Phalloidin Alexa Fluor647 antibody (1:100, Thermo Fisher Scientific) diluted in 0.5% BSA solution overnight at 4 °C.
CD4+ or CD8+ T cells in 3D culture conditions were fixed in 4% PFA for 3 h, permeabilized with 0.3% TRITON X-100 (Sigma-Aldrich) diluted in PBS for 50 min, and incubated in 5% BSA blocking solution for 1 h at RT. The constructs were incubated in Phalloidin Alexa Fluor647 (1:50, Thermo Fisher scientific) dilution in 0.5% BSA solution overnight at 4 °C.
Thereafter, 2D and 3D samples were washed in 1× PBS. Nuclei were counterstained with Dapi (1:1000) for 1 h and washed twice with 1× PBS.
The immunofluorescence analysis was performed after the cell encapsulation in h3D and t3D bulks (0 h).
CD4+ or CD8+ T cells in 3D culture conditions were fixed in 4% PFA for 3 h, permeabilized with 0.3% TRITON X-100 (Sigma-Aldrich) diluted in PBS for 50 min, and incubated in 5% BSA blocking solution for 1 h at RT. The constructs were incubated in Phalloidin Alexa Fluor647 (1:50, Thermo Fisher scientific) dilution in 0.5% BSA solution overnight at 4 °C.
Thereafter, 2D and 3D samples were washed in 1× PBS. Nuclei were counterstained with Dapi (1:1000) for 1 h and washed twice with 1× PBS.
The immunofluorescence analysis was performed after the cell encapsulation in h3D and t3D bulks (0 h).
7
PKH26 Cell Labeling and Sorting Protocol
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PKH26 (Sigma-Aldrich, St. Louis, MO, USA) labeling was performed according to the manufacturer’s protocol. Label-retaining cells were sorted by FACS during which non-viable cells were excluded by propidium iodide staining. Sorted cells were then fixed in suspension for 10 minutes in 10% paraformaldehyde. Equal numbers of PKH26-ve/low and PKH26+ve cells were centrifuged onto slides by spinning for 2 minutes at 1500 rpm in a Cytospin 4 Cytocentrifuge (Thermo Fisher Scientific), or pelleted and resuspended in growth media and allowed to attach to coverslips. Cytospun cells were subsequently immunostained as described in Mani et. al. [4 (link)]. Images were acquired using a Cytation™ 3 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT, USA), and analyzed for fluorescence intensity using Gen5 image analysis software (BioTek).
8
Myeloperoxidase Activity Assay Protocol
9
Immunofluorescence Staining and Analysis
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Cells were grown on coverslips or cytospun (Cytospin 4 Cytocentrifuge; Thermo Fisher Scientific Inc.) onto slides and fixed in 2% PFA and permeabilized with 0.1% Triton-X. Primary antibodies (see Supplementary Table S1 ) were diluted in blocking buffer (20% horse serum, 0.1% FBS, 0.03% sodium azide, in PBS) and incubated overnight at 4 °C in a humidifying chamber. Secondary antibodies (Alexas 488 and 594; Life Technologies) were applied and nuclei were counterstained with DAPI. Images were taken with an Axio Imager M1 microscope (Zeiss, Toronto, ON, Canada) and analyzed using ImageJ software. Positively stained cells were scored as indicated, relative to DAPI-stained nuclei.
10
BALF Collection and Cell Analysis
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To obtain BALF, the right lung was flushed three times with 0.4 ml 2 mM EDTA in PBS. The total cell number was determined using the Cedex HiRes automated cell analyzer (Roche, Basel, Switzerland). To determine the cell types, cytospin slides were made with the CytoSpin 4 Cytocentrifuge (Thermo Fisher Scientific) and stained with May-Grünwald/Giemsa (Merck). Cell types were determined using standard microscopic criteria and counted in a blinded manner at 1000× magnification (Axiovert 40 CFL, Zeiss, Oberkochen, Germany).
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