To sort Gr-1high an Gr-1int cells, single cell suspensions were prepared from M. tuberculosis Erdman infected lungs of mice as described above. After incubating with 5ug/ml of Fc-Block (eBiosciences) for 15 minutes at 4°C, cells were stained with anti-CD11b (clone M1/70) and Gr-1(clone RB6-8C5) as described above. A live-dead stain was used to exclude the dead cells. Cells were then fixed with Cytofix (BD biosciences). Fixed cells were further fractionated into CD11b+Gr1high and CD11b+Gr-1int by a FACS Aria IIu Cell sorter (BD biosciences). Sorted cells were analyzed for purity using FACS Aria and both the Gr-1high and Gr-1int cells were obtained at >90% purity. FACS sorted cells were cytospun onto cytoslides (Thermo-scientific), stained with DAPI and images were acquired in a Delta Vision deconvolution microscope. The images were taken with a 60× objective and the Delta Vision SoftWorx software was used to deconvolve the images.
Cytoslides
Manufactured by Thermo Fisher Scientific
Cytoslides are specialized glass microscope slides designed for the preparation and examination of cellular samples. They provide a standardized and consistent platform for the mounting and analysis of cells, tissues, or other biological materials.
Automatically generated - may contain errors
Lab products found in correlation
Fc block, by Thermo Fisher Scientific (2 mentions)
Facsaria iiu cell sorter, by BD (2 mentions)
Cytofix, by BD (2 mentions)
Cytofunnel, by Thermo Fisher Scientific (2 mentions)
Cytospin 4 cytocentrifuge, by Thermo Fisher Scientific (2 mentions)
Anti lc3, by Cell Signaling Technology (1 mentions)
Fv1000 d ix81 confocal microscope, by Olympus (1 mentions)
Alexa fluor 594 anti rabbit igg antibodies, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent with dapi, by Thermo Fisher Scientific (1 mentions)
Cytospin 4, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
May gr nwald and giemsa stain, by Merck Group (1 mentions)
6 protocols using cytoslides
1
Sorting Gr-1high and Gr-1int Cells from M. tuberculosis Infected Lungs
2
Sorting Gr-1high and Gr-1int Cells from M. tuberculosis Infected Lungs
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To sort Gr-1high an Gr-1int cells, single cell suspensions were prepared from M. tuberculosis Erdman infected lungs of mice as described above. After incubating with 5ug/ml of Fc-Block (eBiosciences) for 15 minutes at 4°C, cells were stained with anti-CD11b (clone M1/70) and Gr-1(clone RB6-8C5) as described above. A live-dead stain was used to exclude the dead cells. Cells were then fixed with Cytofix (BD biosciences). Fixed cells were further fractionated into CD11b+Gr1high and CD11b+Gr-1int by a FACS Aria IIu Cell sorter (BD biosciences). Sorted cells were analyzed for purity using FACS Aria and both the Gr-1high and Gr-1int cells were obtained at >90% purity. FACS sorted cells were cytospun onto cytoslides (Thermo-scientific), stained with DAPI and images were acquired in a Delta Vision deconvolution microscope. The images were taken with a 60× objective and the Delta Vision SoftWorx software was used to deconvolve the images.
3
Preparation of FISH-ready Samples
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Thin-layered samples on a cover-slide suitable for FISH were obtained with a Cytospin protocol. 150 μl of the sample were deposited in a Cytofunnel (1102548; Thermo Fisher Scientific). Samples were then centrifuged at 72g for 10 min at room temperature with a Cytospin 4 cytocentrifuge (Thermo Fisher Scientific) on Cytoslides (5991059; Thermo Fisher Scientific). Cells were fixed in PBS-PFA 4% for 30 min and then conserved frozen in 70% ethanol at −20°C. CoronaFISH labeling was performed with Cy3-labeled plus-strand probes as described above, with one exception: for hybridization 400 μl hybridization buffer was used per sample instead of 100 μl (with the same final probe concentration).
4
Immunofluorescence Assay for Autophagy
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Cells were treated with or without 300 nM 17-AAG and/or 100 nM BAFA1 for 6 h, and 1 × 106 cells were collected into 1.5 mL microcentrifuge tubes by centrifugation at 200 × g for 5 min at 4° C. After washing cells with ice-cold PBS, they were fixed with 3.7% formalin/PBS for 15 min at room temperature, permeabilized with 0.2% Triton X-100/PBS for 15 min at room temperature, transferred to FBS-coated 0.2-mL PCR tubes, and then blocked in 3% BSA/PBS for 30 min at room temperature. Cells were incubated with anti-FLAG M2 (50 μg/mL, Sigma-Aldrich) and anti-LC3 (1:50 dilution, Cell Signaling Technology) antibodies overnight at 4° C, followed by incubation with secondary antibodies, Alexa Fluor® 488 goat anti-mouse IgG and Alexa Fluor® 594 anti-rabbit IgG antibodies (Invitrogen), for 2 h at room temperature. Cells were washed three times with PBS, plated on cytoslides (Thermo Fisher Scientific Inc., Waltham, MA) using Cytospin®4 (Thermo Fisher Scientific Inc.), and mounted with Prolong® Gold antifade reagent with DAPI (Invitrogen). Acquisition of images was performed using a FV1000-D IX81 confocal microscope (Olympus Corp., Tokyo, Japan). Confocal 2-D TIFF images were merged using Adobe® Photoshop CC software (Adobe Systems Inc., San Jose, CA).
5
Cytological Examination of Hematopoietic Cells
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Mouse BM, spleen, thymic, or nodal tissue was dissociated into a single-cell suspension. Cells were spun onto Cytoslides (Thermo Scientific) and stained with May-Grünwald and Giemsa stains (Sigma). In some experiments, cells were purified by FACS before spinning onto slides and staining. Cell morphology was examined for stage of differentiation arrest and lymphoid or myeloid morphology blindly. For examination of tissue sections, fresh tissue was fixed in 4% paraformaldehyde overnight and dehydrated with 70% ethanol before embedding and staining with H&E by standard techniques.
6
Cytospin-based FISH Sample Preparation
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Thin-layered samples on a cover-slide suitable for FISH were obtained with a Cytospin protocol.
150 µL of the sample were deposited in a Cytofunnel (Thermo Scientific 1102548). Samples were then centrifuged (800 rpm, 10 min, room temperature) with a Cytospin 4 cytocentrifuge (Thermo Scientific) on Cytoslides (Thermo Scientific 5991059). Cells were fixed in PBS-PFA 4%
for 30 min and then conserved frozen in 70% ethanol at -20°C. FISH protocol was performed with Cy3-labeled plus-strand probes labeled with as described above, with one exception: for hybridization 400 µL hybridization buffer was used per sample instead of 100 µL (with the same final probe concentration). and stored until use.
150 µL of the sample were deposited in a Cytofunnel (Thermo Scientific 1102548). Samples were then centrifuged (800 rpm, 10 min, room temperature) with a Cytospin 4 cytocentrifuge (Thermo Scientific) on Cytoslides (Thermo Scientific 5991059). Cells were fixed in PBS-PFA 4%
for 30 min and then conserved frozen in 70% ethanol at -20°C. FISH protocol was performed with Cy3-labeled plus-strand probes labeled with as described above, with one exception: for hybridization 400 µL hybridization buffer was used per sample instead of 100 µL (with the same final probe concentration). and stored until use.
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