NOD.CB17-Prkdcscid/J (NOD/SCID) mice (male, eight weeks, from Dr. K. Whartenby) were injected (i.v.) with 3×107 aAPC or aAPCCD47+. After 1h animals were sacrificed and organs harvested. Single cell suspensions were generated utilizing a 70 µm cell strainer (Fisherbrand). Beads were isolated on a magnet and washed with PBS. Recovered beads were counted and percentage calculated (% = amount organ-specific beads x 100/ amount recovered beads).
70 m cell strainer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The 70 µm cell strainer is a laboratory equipment used for mechanical separation and filtration of cell suspensions. It features a nylon mesh with a pore size of 70 micrometers, designed to remove larger cell aggregates, debris, and clumps while allowing the passage of single cells.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Merck Group (5 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (4 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (4 mentions)
Liberase, by Roche (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Facs diva, by Tree Star (2 mentions)
Pdgfrα cd140a apc, by Thermo Fisher Scientific (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Trypsin edta, by Thermo Fisher Scientific (2 mentions)
Lsr 2, by BD (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Collagenase, by Merck Group (2 mentions)
Live dead fixable aqua dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Collagenase d, by Merck Group (2 mentions)
Rbc lysis buffer, by BioLegend (2 mentions)
Lsrfortessa, by BD (2 mentions)
Liberase tl, by Roche (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640, by Corning (2 mentions)
Percoll, by GE Healthcare (2 mentions)
40 protocols using 70 m cell strainer
1
Biodistribution of Targeted Beads in Mice
2
Isolation and Characterization of Murine Osteoblasts
3
3D Colonoid Harvesting and Cultivation
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The 3D colonoids were harvested from Matrigel after being cultured for 4–6 days. This was achieved by dissolving the Matrigel dome using Cell Recovery Solution (Corning), following the manufacturer's instructions. The collected organoids, along with the solution, were then treated by incubating them in TrypLE Express containing 10 µM of Y-27632 for 10 min at 37 °C. Subsequently, the disrupted organoids were filtered through a 70 µm cell strainer (Fisher Scientific) and centrifuged at 200 g, 4 °C for 5 min.
For the preparation of Falcon cell culture inserts (0.4 µm pores, Falcon), a coating process was carried out using 100 µg/ml Matrigel and 30 mg/ml collagen I (Gibco) in DMEM/F12 supplemented with 2 mM GlutaMAX, 10 mM HEPES, and 1 × Penicillin/Streptomycin. This coating process took place at 37 °C for 1 h. Dissociated single cells obtained from the colonoids were then quantified using a Hemocytometer and seeded at a concentration of 106 cells/ml within the pre-coated cell culture insert. The culture medium was changed every other day.
For the preparation of Falcon cell culture inserts (0.4 µm pores, Falcon), a coating process was carried out using 100 µg/ml Matrigel and 30 mg/ml collagen I (Gibco) in DMEM/F12 supplemented with 2 mM GlutaMAX, 10 mM HEPES, and 1 × Penicillin/Streptomycin. This coating process took place at 37 °C for 1 h. Dissociated single cells obtained from the colonoids were then quantified using a Hemocytometer and seeded at a concentration of 106 cells/ml within the pre-coated cell culture insert. The culture medium was changed every other day.
4
Isolation and Purification of Rat Mesenchymal Stromal Cells
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After humane euthanasia, Lewis rat femurs were removed, cleaned of all muscle and adherent tissue, and the epiphyses removed using a bone rongeur. Bone marrow (BM) was flushed from the bone shafts into a 50 mL conical tube using a 10 cc syringe fitted with an 18-gauge needle and filled with 10 mL ice cold HBSS containing 5% FBS. BM cells were collected by centrifugation at 450× g for 5 min at 4 °C and the pellet re-suspended in 45 ml of alpha-MEM containing 20% FBS and 1% penicillin/streptomycin (= growth media). The re-suspended cells were seeded (1–1.5 × 108 cells/dish) into three 100 mm tissue culture dishes containing 15 ml of growth media. Half media changes were performed on day 3 and day 5. After 7 days, passage 1 (P1) BM cells were detached by treating with collagenase type II (400 units/ml) (Worthington, Lakewood, NJ, USA) for 5–10 min at room temperature. The harvested cells, mainly containing MSCs (> 90% CD90 shown in Fig. 6 A), were filtered through a 70 µm cell strainer (Fisher Scientific, cat. # 22363548, Fair Lawn, NJ, USA) to create a single cell suspension for subsequent experiments.
5
Tumor Dissociation and Flow Cytometry
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Mice were euthanized by CO2 asphyxiation and tumors were manually extracted. Tumors were minced with a razor blade and resuspended in dissociation buffer: Hank’s balanced salt solution (HBSS) supplemented with 0.1 mg/mL Liberase TL (Sigma Aldrich, 05401020001) and 0.1 mg/mL DNAse I (Sigma Aldrich, 10104159001). Resuspended tumors were incubated in 15 mL conical tubes at 37 °C for 45 min, with gentle agitation every 15 min. Tumors were then passed through a 70 µM cell strainer (Fisher Scientific, 08-771-2) and red blood cells were lysed with ACK Lysis Buffer (Fisher Scientific, A1049201). Single cell suspensions were incubated with Fc block (BD Biosciences, 553142) and a fixable live/dead aqua stain (Thermo Fisher, L34957). Cells were then stained with a T cell panel: CD45, CD3, CD4, CD8 or a myeloid cell panel: CD45, CD11b, Ly6G, Ly6C, CD11c, and F4/80 of fluorescent antibodies. Stained cells were analyzed on a BD Biosciences LSR II flow cytometer (Stanford FACS Facility).
6
Isolation of Murine Skin and Spleen Cells
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Murine skin was minced into small pieces (2 mm3) and digested in 500 µg/ml Liberase DH Research Grade (Roche) for 30 min at 37°C. One hundred mM EDTA was added and tissue was processed into a single-cell suspension by repeated pipetting. Cells were then passed through a 70 µm cell strainer (Fisher). Total skin cells were analyzed. Spleens were processed into a single cell suspension by passage through a 70 µm cell strainer. CD25+ T cells were removed, where indicated, using magnetic beads, as described by the manufacturer (Invitrogen). Bead based depletion was 90–95% effective, as measured by FoxP3 staining.
7
3D Agarose Culturing of 3T3-L1 Cells
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The 3T3-L1 cells were grown to 70% confluence under standard monolayer conditions
as recommended by the manufacturer and collected via a 5-min incubation at
37oC with 1× trypsin ethylenediaminetetraacetic acid (EDTA;
ATCC). Cells were filtered through a single-use sterile 70 µm cell strainer
(Fisher Scientific) to remove cell clumps. Single cells were counted with a
hemocytometer, pelleted and resuspended at a concentration of
5 × 105 cells/mL in 0.5% low-temperature agarose in DMEM. The 0.5%
low-temperature agarose (Bio-Rad) was created by mixing equal volumes of 1%
low-temperature agarose with 2× DMEM. A 10 μL drop of individual cells suspended
in 0.5% low-temperature agarose was plated in the center of a 35-mm cell culture
dish previously coated with a 1% high-temperature agarose (Bio-Rad). Cultures
were allowed to gel at 4oC for 15 minutes prior to feeding with 2 mL
of media (DMEM, 10% fetal bovine serum [FBS] or 10% BCS, 0.1% pen-strep). Cells
were fed by complete media change of 2 mL at half-week intervals. A detailed
description of the protocol for 3D agarose is described in Kinder and Aulthouse.3 (link)
as recommended by the manufacturer and collected via a 5-min incubation at
37oC with 1× trypsin ethylenediaminetetraacetic acid (EDTA;
ATCC). Cells were filtered through a single-use sterile 70 µm cell strainer
(Fisher Scientific) to remove cell clumps. Single cells were counted with a
hemocytometer, pelleted and resuspended at a concentration of
5 × 105 cells/mL in 0.5% low-temperature agarose in DMEM. The 0.5%
low-temperature agarose (Bio-Rad) was created by mixing equal volumes of 1%
low-temperature agarose with 2× DMEM. A 10 μL drop of individual cells suspended
in 0.5% low-temperature agarose was plated in the center of a 35-mm cell culture
dish previously coated with a 1% high-temperature agarose (Bio-Rad). Cultures
were allowed to gel at 4oC for 15 minutes prior to feeding with 2 mL
of media (DMEM, 10% fetal bovine serum [FBS] or 10% BCS, 0.1% pen-strep). Cells
were fed by complete media change of 2 mL at half-week intervals. A detailed
description of the protocol for 3D agarose is described in Kinder and Aulthouse.3 (link)
8
Murine Lung Cell Isolation
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The left lung of euthanized mice was collected in 700 µL sterile PBS and kept on ice for further processing. The PBS was decanted, and the tissue was minced manually with dissection scissors. Minced tissue was resuspending in 2 mL IMDM (Gibco) containing 2 mg/mL collagenase (Sigma-Aldrich) and 80 U/mL DNase1 (Sigma-Aldrich) and incubated at 37 °C with shaking at 233 rpm for 1 h. Digested tissue was passed through a 70 µm cell strainer (Fisher) and red blood cells were removed using ACK lysis buffer (Gibco). Isolated cells were resuspended in 1 mL IMDM containing 10% FBS (Hyclone) and counted on a Cellometer for downstream applications.
9
Lung Immune Cell Isolation and Analysis
10
Isolation and Characterization of Murine Osteoblasts
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Femurs, tibias, iliac crests and humeri were carefully dissected and gently crushed in 5ml Ca2+, Mg2+-free HBSS (Sigma) with a pestle and a mortar (Coors Tek), then supernatants were filtered through a 70µm cell strainer (Fisher) into a 50ml tube on ice. Bone fragments were crushed for two additional times in 5ml HBSS and supernatants were filtered into the same tube. Tissue remnants were incubated with 2 Wunsch units of Liberase TM (Roche) at 37°C for 45 minutes on a shaking incubator (Thermomixer, Eppendorf). 0.25% trypsin-EDTA (Gibco) was added for experiments to harvest osteoblasts. Cells were mechanically triturated using an 18-gauge needle and a 1ml syringe (BD) and filtered into the same tube. Cells were pelleted, resuspended and layered over Ficoll-Paque PLUS (GE Healthcare) to collect low-density cell fractions. Cells were stained with anti-mouse CD45-APC, CD45-eFlour 450, Ter119-eFlour 450, Sca1-Alexa Flour 700 (1:500, eBioscicence), PDGFRα (CD140a)-APC (1:250, eBioscience) or their isotype controls (1:500, eBioscience) in DPBS/2%FBS on ice for 30 min. Flow cytometry was performed using a four-laser BD LSRII (Ex.355/407/488/633nm) and FACSDiva, and analyzed on FlowJo (TreeStar). Representative plots of at least three independent biological samples were shown in the figures.
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