Total and differential cell counts have been performed according to the literature [62 (link),73 (link)]. Briefly, BALf pellets were resuspended in 500 μL of DMEM (10% FBS, 1% penicillin-streptomycin, 1% glutamine), and total cell counts performed with a Burker chamber, using the Trypan Blue exclusion method. A cell aliquot (240,000 cells, 800 cells/μL) was smeared in duplicate onto slides using StatSpin Cytofuge 2 (Beckman Coulter Brea, CA, USA) 40× g for 7 min at room temperature. Subsequently, the smears were stained with Diff Quik (Medion Diagnostic, Miami, FL, USA) for cell differential count, according to the manufacturer’s instructions. Macrophages, polymorphonuclear leukocytes (PMNs) and lymphocytes were identified by their characteristic shapes.
Statspin cytofuge 2
Manufactured by Beckman Coulter
Sourced in United States
The StatSpin Cytofuge 2 is a centrifuge designed for the preparation of cellular samples for microscopic examination. It is capable of processing multiple samples simultaneously and provides a consistent, reproducible method for concentrating cells onto slides.
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Lab products found in correlation
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Hema3 stain set, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Lsm 710 nlo confocal microscope, by Zeiss (1 mentions)
Biorevo bz7000, by Keyence (1 mentions)
May grunwald giemsa, by Merck Group (1 mentions)
Shandon kwik diff stain, by Thermo Fisher Scientific (1 mentions)
Evos fl auto imaging system, by Thermo Fisher Scientific (1 mentions)
In situ cell death detection fluorescein, by Roche (1 mentions)
Las x software, by Leica (1 mentions)
Dmi8 inverted fluorescence microscope, by Leica (1 mentions)
Vectashield antifade mounting medium with dapi, by Vector Laboratories (1 mentions)
Recombinant dnase 1, by Roche (1 mentions)
Dm2500, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Axiocam mrc5 camera, by Zeiss (1 mentions)
Zen blue edition software, by Zeiss (1 mentions)
Axiovert 200 microscope, by Zeiss (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Fluorescently labeled secondary antibody, by Jackson ImmunoResearch (1 mentions)
10 protocols using statspin cytofuge 2
1
Differential Cell Count Protocol
2
Murine Pulmonary Function and Lavage
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After completion of the pulmonary function testing, mice were euthanized with an overdose of ketamine/xylazine, and blood was collected via cardiac puncture21 ,22 (link). In a subset of mice, bronchoalveolar lavage (BAL) was performed, and lungs were harvested and placed in RNAlater® (Life Technologies, Burlington, ON, Canada) for storage at −20 °C, or inflation-fixed to 25 cm H2O in 4% paraformaldehyde for subsequent histologic analysis27 (link). BAL fluid was centrifuged (400 RPM, 10 min) to obtain cell-free supernatants and stored at −80 °C; the cell pellet was re-suspended in 500 ml phosphate buffered saline (PBS) for total leukocyte counts using a hemocytometer21 –23 (link). Differential leukocyte counts were performed using a StatSpin Cytofuge 2 (Beckman Coulter Inc, CA, USA), with 70 μl of the resuspended pellets stained using the HEMA3 stain set (Fisher Scientific, PA, USA).
3
Imaging Cells with DAPI Staining
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Samples were spun onto poly-l-lysine slides using Stat Spin CytoFuge 2 (Beckman Coulter; Brea, CA USA) set at 800 rpm for 4 minutes. One drop of mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI) stain to visualize nuclear structures (Vectashield, Vector Laboratories, Burlingame, CA, USA) was placed onto the glass slide before adding glass coverslip and sealing with nail polish. Slides were imaged using a fluorescence light microscope (BIOREVO BZ7000; Keyence; Osaka, Japan) and a Zeiss LSM 710 NLO confocal microscope.
4
BAL Leukocyte Quantification in Mice
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Bronchoalveolar lavage (BAL) was performed utilizing euthanized mice as previously described [16 (link)]. In brief, after tracheal intubation, the mice were instilled with sterile cold PBS (0.8 mL) and flushed (×3). The total BAL leukocyte concentration was determined immediately using a hemocytometer. After centrifugation (1000 ×g; 10 min, 4°C), the cell-free BAL fluid was then aliquoted and stored (−80°C) for further analyses (see below). The cell pellet was then re-suspended in PBS and underwent careful centrifugation (600 rpm, 4 min) in a designated centrifuge (StatSpin® Cytofuge 2, Beckman Coulter Inc,™, Indianapolis, IN) The cytospin slides were then stained (May-Grunwald- Giemsa™, Sigma-Aldrich Co. LLC., MO) and the cell differential counts were performed in a blinded manner by an experienced operator. The operator counted 300 leukocytes (i.e. macrophages, neutrophils & lymphocytes) per slide, all identified by standard morphology using light microscopy (×400 magnification; Olympus Optical™, Tokyo, Japan).
5
Cell Morphology and Tissue Histology
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To assess cell morphology after antibody treatment, cells were spun onto glass slides (5 min at 600 rpm) using a StatSpin CytoFuge 2 (Beckman Coulter) and May-Grünwald Giemsa stained or stained with a modified Giemsa stain (Shandon Kwik-Diff Stains; Thermo Scientific). Femurs and liver sections from mice were fixed in 10% neutral buffered formalin, decalcified (for femurs), and paraffin embedded. Femurs and livers were sectioned and H&E–stained for histological analysis. Images were captured at room temperature on an EVOS FL Auto Imaging System with a color high-sensitivity CMOS, 1/2” 2,048 × 1,536, 3.1 megapixel camera (Life Technologies), exported as TIFF files, and images were processed in Adobe Photoshop.
6
In Situ Cell Death Detection
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The In Situ Cell Death Detection Fluorescein (Roche) kit was used to detect cell death after 24‐hour propranolol treatment (0‐200 μmol/L).
3‐5 × 104 cells were transferred with a cytocassette to be spun in a cytocentritifugue (StatSpin Cytofuge 2; Beckman coulter). Manufacturer's instructions were followed. 50 UI of DNase I recombinant (Roche) was used as a positive control. Vectashield Antifade Mounting Medium with DAPI (Vector laboratories) was used to identify nucleus. Images were captured using the Leica DMi8 inverted fluorescence microscope and the Leica LAS X software.
3‐5 × 104 cells were transferred with a cytocassette to be spun in a cytocentritifugue (StatSpin Cytofuge 2; Beckman coulter). Manufacturer's instructions were followed. 50 UI of DNase I recombinant (Roche) was used as a positive control. Vectashield Antifade Mounting Medium with DAPI (Vector laboratories) was used to identify nucleus. Images were captured using the Leica DMi8 inverted fluorescence microscope and the Leica LAS X software.
7
Characterizing BMDC Nanoparticle Uptake
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Bone marrow-derived cells (BMDCs) were freshly isolated from female B6 mice and cultured in RPMI media containing 0.2 mg/mL Rhod-NPs. After 24 hr of incubation, the cells were filtered with a 20 µm strainer, fixed with 10% buffered formalin, and adhered onto a poly-lysine-coated glass slide using StatSpin CytoFuge 2 cytocentrifuge (Beckman Coulter, Brea, CA, USA) for 10 min at 1,200 rpm then counterstained with DAPI (Vector Laboratories, Newark, CA, USA). Immunofluorescence staining was performed following incubation with Rhod-NPs for 24 hr. The cells were stained for CD3, CD45, or Ly6G with Alexa 647-labeled antibody for 1 h at RT, followed by filtration, fixation, and transfer to a glass slide. The cells were then counterstained with DAPI and imaged using a Leica DM 2500 (Leica Biosystems, Nussloch, Germany) fluorescence microscope with a 40× objective.
8
Cytological Staining Protocol for Microscopy
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Samples were cytospun on microscopic slides (1000 rpm, 2 min) using StatSpin Cytofuge 2 (BeckmanCoulter, Marseille, France) and left to dry overnight. Slides were stained with May-Grünwald stain (50% working solution, 5 min) and, subsequently, Giemsa stain (10% working solution, 20 min). Morphology was examined using AxioVert 200 microscope and images were obtained using AxioCam MRc 5 camera and ZEN software, blue edition (Carl Zeiss AG, Oberkochen, Germany).
9
Immunostaining Bacteria for Microscopy
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Immunostaining of bacteria for fluorescence microscopy was carried out essentially as described above for flow cytometry, with the exception that the Alexa 555-conjugated goat anti-mouse antibody (1:1.000, Cell Signaling) was used to detect anti-flag antibodies. Stained cells were fixed on microscope slides coated with poly-L-lysine (Sigma-Aldrich) using a cytocentrifuge StatSpin Cytofuge 2 (Beckman Coulter, Brea, CA, USA). The prepared samples were examined with LSM 710 confocal microscope (Carl Zeiss, Oberkochen, Germany). The images were analyzed and processed with Image J version 1.52a [59 (link)].
10
Identifying Endometrial Epithelial Cells
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Sloughed-off endometrial cells in uterine secretions were collected into 300 mL PBS and sedimented onto slides using a StatSpin CytoFuge 2 (Beckman Coulter) at 1,000 rpm for 5 minutes. Cells were fixed at room temperature with 4% paraformaldehyde for 15 minutes and permeabilized with 1% Triton X-100 for 5 minutes. After blocking with 1% dried milk in PBS for 15 minutes, EECs were stained with rabbit antibodies against cytokeratin 8 (diluted 1:200; Thermo Scientific/Lab Vision) and NCSs and nuclear pore complexes with mouse monoclonal antibody (mAb414 diluted 1:5,000; Covance Research Products) against a subset of nuclear pore complex proteins that are enriched in NCSs. Fluorescently labeled secondary antibodies (Jackson Immunoresearch) were used to detect the rabbit (rhodamine labeled goat anti-rabbit at 1:200) and mouse antibodies (DyLight488 goat anti-mouse at 1:500). Primary antibodies were incubated for 60 minutes and secondary antibodies for 30 minutes in blocking buffer at room temperature. Nuclei were counterstained with the DNA stain 4',6-diamidino-2-phenylindole (Sigma).
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