When indicated, mice were challenged on three consecutive days with 0.5 µg/mouse in 50 µl of carrier-free rmIL-33 or rmIL-25 (BioLegend) diluted in PBS. On day 4, lungs were collected and processed to single cell suspensions for the indicated readout. Briefly, following transcardial perfusion with PBS 1× to clear lungs of red blood cells, collected lungs were then digested in collagenase Type IV (400 U/ml) at 37°C for 1 h and processed to single cell suspension through a 70-mm nylon cell strainer (Falcon) as described previously (20 (link), 21 (link)). For experiments involving bone marrow cells, cells were isolated by flushing the bone with 5 ml ice cold PBS 1× from one tibia using a 25-G syringe (Becton Dickinson), red blood cells were lysed and cells used for the indicated experiment. For experiments involving peripheral blood, 2 ml of blood was collected via heart puncture using a 27-G needle syringe (Becton Dickinson) and kept in PBS 1% EDTA, red blood cells were lysed, and peripheral blood cells used for the indicated experiment. In experiments involving in vivo antibody blocking antibodies, 100 µg anti-ICAM-1 (YN1/1.7.4, BioXcell), anti-ICAM-2 (3C4, Biolegend) or corresponding isotype controls were injected through the tail-vein on days 1, 2, and 3 prior rmIL-33 intranasal challenge.
70 mm nylon cell strainer
Manufactured by BD
Sourced in United States
The 70-mm nylon cell strainer is a laboratory equipment used for filtering and separating cells or other biological materials. It features a nylon mesh with a pore size of 70 micrometers, which allows the passage of cells or particles smaller than the pore size while retaining larger ones.
Automatically generated - may contain errors
Lab products found in correlation
Hepes, by Thermo Fisher Scientific (2 mentions)
Pharm lysing buffer, by BD (2 mentions)
Cd16 cd32 fc block antibody, by BD (2 mentions)
Collagenase d, by Roche (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
25 g syringe, by BD (1 mentions)
Rmil 25, by BioLegend (1 mentions)
Rmil 33, by BioLegend (1 mentions)
Dnaase type 1, by Merck Group (1 mentions)
Collagenase type 2, by Merck Group (1 mentions)
Ficoll paque plus solution, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Facsaria 3, by BD (1 mentions)
Ca2 mg2, by Corning (1 mentions)
Intesticult organoid growth medium, by STEMCELL (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Gemini Bio (1 mentions)
Matrigel, by Corning (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
16 protocols using 70 mm nylon cell strainer
1
Intranasal IL-33/IL-25 Challenge in Mice
2
Isolation of Hepatic Lymphocytes
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Hepatic lymphocytes were isolated from the liver as described previously [13 (link)] with some modifications. Briefly, livers were perfused with pre-warmed PBS to flush blood from the hepatic vasculature and were forced through a 70 mm nylon cell strainer (BD Falcon, Franklin Lakes, NJ). After washing, cell pellets were suspended in 5 ml of pre-warmed Ca2+/Mg2+-free HBSS supplemented with 10% FBS, 0.05% Collagenase type II (Sigma) and 500 U/ml DNAase type I (Sigma) and digested at 37°C for 30 minutes. Cells were then layered onto 40% Percoll solution (Axis-Shield) in RPMI 1640 for density separation, and centrifuged at 2000 g for 15 minutes at 4°C without brakes. Cell yields and viabilities were determined by trypan blue exclusion microscopy.
3
Isolation and Sorting of Tumor-Infiltrating Lymphocytes
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Four tumor samples were prepared as previously described [21 (link)]. In brief, the freshly obtained surgical tumor samples were cut into approximately 1 mm3 pieces and incubated in DMEM + collagenase IV (1 mg/ml) + DNase (15 μg/ml) for 20 to 40 min at 37 °C. After tissue digestion, cell suspensions were subsequently passed through a 70-mm nylon cell strainer (Falcon). The remaining tissue pieces were mechanically dissociated using the back of a syringe over the cell strainer. All the cell samples were subsequently centrifuged for 10 min at 400 g. After centrifugation, cells were resuspended in 1 × phosphate-buffered saline (Invitrogen) with 1% fetal bovine serum. From single-cell suspensions, lymphocytes were isolated using Ficoll-Paque PLUS solution (Sigma-Aldrich).
Lymphocytes were stained with the following antibodies: APC/Cy7 anti-human CD45 (HI30), PE anti-human CD3 (OKT3), PE/Cy7 anti-human CD4 (OKT4), and PerCP/Cy5.5 anti-human CD8 (SK1) from Biolegend. CD4 + T cells and CD8 + T cells were sorted using the FACSAria III (BD).
Lymphocytes were stained with the following antibodies: APC/Cy7 anti-human CD45 (HI30), PE anti-human CD3 (OKT3), PE/Cy7 anti-human CD4 (OKT4), and PerCP/Cy5.5 anti-human CD8 (SK1) from Biolegend. CD4 + T cells and CD8 + T cells were sorted using the FACSAria III (BD).
4
Intestinal Organoid Isolation and Culture
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Intestines were washed in ice-cold PBS (Mg2+/Ca2+ (Corning, cat # 21-031-CM), containing 2% BSA (Gemini Bio-products, cat #900–208) and 2% antibiotic-antimycotic (Gibco, cat #15240–062). Crypts and villi were exposed by dicing the intestines into small pieces (1–2 cm long), followed by extensive washes to remove contaminants. Then, a gentle cell dissociation reagent (Stem cell technologies, cat #7174) was used according to the manufacturer’s instructions. Briefly, intestinal pieces were incubated on a gently rotating platform for 15 min. After that, the gentle cell dissociation reagent was removed and the intestines were washed 3 times with a PBS wash buffer with vigorous pipetting. The first and second fractions that usually contain loose pieces of mesenchyme and villi were not used. Fractions three and four containing the intestinal crypts were collected and pooled. Isolated crypts were filtered through a 70mm nylon cell strainer (Falcon, cat #352350). Crypts were counted, then embedded in Matrigel (Corning, growth factor reduced, cat #354230), and cultured in Intesticult organoid growth medium (Stem cell technologies, cat #6005). For mouse colon organoids, additional Wnt3a (300 ng/μL, R&D, cat #5036-WN-010) was added. Intestinal organoids used in this study were generated from WT and APCmin/+ mice at 37°C, 5% CO2.
5
Isolation and Sorting of E10.5 Cardiac Cells
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E10.5 double transgenic hearts were dissected, pooled and digested with 0.25% Trypsin/EDTA (Invitrogen), neutralized in DMEM (Invitrogen) containing 5% FBS and 10 mmol l−1 HEPES (Invitrogen), rinsed and resuspended in phosphate-buffered saline (PBS) and passed through a 70-mm nylon cell strainer (Falcon). Samples were sorted on a FacsAria flow cytometer (BD) using Influx software. Samples were gated to exclude debris and cell clumps. The number of E10.5 AV canal GFP cells and chambers–Katushka cells per embryo obtained were typically 600–900 and 2,400–3,600, respectively. Fluorescent cells were collected into PBS and processed for RNA extraction or ChIP-seq.
6
Isolation of Astrocytes from Neonatal Rats
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The procedure for
isolating the astrocytes from neonatal rats was approved by the Institutional
Animal Care and Use Committee (IACUC) and completed at Wichita State
University in Wichita, Kansas.42 (link),43 (link) In brief, cerebral
cortexes were isolated from the brains of neonatal rats (postnatal
day P1-2 rats) after they were sacrificed. The cortex tissues were
triturated gently through a 5 mL syringe with a needle. The tissue
suspension was passed through a 70 mm nylon cell strainer (BD Falcon,
Durham, NC), and the flow-through was collected with a 50 mL conical
tube. The isolated cells were cultured for about 7–14 days.
After reaching confluency, the cultures were shaken to remove macrophages
and progenitor cells. The adherent astrocytes were cultured subsequently
for cell–nanofiber interaction. The cells were grown in Dulbecco’s
modified Eagle medium supplemented with 10% fetal bovine serum and
1% penicillin/streptomycin (Life Technologies, Grand Island, NY).
The medium was changed every 2–3 days, and the cell culture
was incubated with 5% CO2 at 37 °C.
isolating the astrocytes from neonatal rats was approved by the Institutional
Animal Care and Use Committee (IACUC) and completed at Wichita State
University in Wichita, Kansas.42 (link),43 (link) In brief, cerebral
cortexes were isolated from the brains of neonatal rats (postnatal
day P1-2 rats) after they were sacrificed. The cortex tissues were
triturated gently through a 5 mL syringe with a needle. The tissue
suspension was passed through a 70 mm nylon cell strainer (BD Falcon,
Durham, NC), and the flow-through was collected with a 50 mL conical
tube. The isolated cells were cultured for about 7–14 days.
After reaching confluency, the cultures were shaken to remove macrophages
and progenitor cells. The adherent astrocytes were cultured subsequently
for cell–nanofiber interaction. The cells were grown in Dulbecco’s
modified Eagle medium supplemented with 10% fetal bovine serum and
1% penicillin/streptomycin (Life Technologies, Grand Island, NY).
The medium was changed every 2–3 days, and the cell culture
was incubated with 5% CO2 at 37 °C.
7
Single-cell Lung Immune Profiling
8
Antigen-Specific T Cell Activation
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Mice were immunized s.c. with 50 μg of OVA-derived SIINFEKL peptide (OVA-1). The SIINFEKL peptide was co-administered with PBS, Hp91 (250 μg), or scrambled Hp91 (250 μg). Peptides were re-suspended in PBS for all immunizations. Mice were boosted two weeks later and spleens and blood were collected one week after the final immunization. Single cell suspensions of splenocytes were prepared by mechanical disruption and separation through a 70 mm nylon cell strainer (BD Biosciences). Red blood cells were lysed using ammonium chloride buffer (Roche Diagnostics, Indianapolis, IN) and the splenocytes were subsequently re-suspended in RPMI 1640 medium (Invitrogen) supplemented with 10 mM HEPES (Invitrogen), penicillin (100 U/ml), streptomycin (100 μg/ml), L-glutamine (2 mM) (Invitrogen), and 5% (vol/vol) fetal calf serum (Omega).
9
Flow Cytometry Analysis of Immune Cells
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For flow cytometry experiments mice were euthanized on days 0, 4 or 7 p. i. and spleens and lungs were collected for analysis. Single cell suspensions were prepared by cutting tissues into small fragments followed by enzymatic digestion for 45 min at 37°C with Collagenase D (2mg/ml) (Roche) in RPMI-1640 medium. Tissue fragments were further disrupted by passage through a 70-mm nylon cell strainer (BD Biosciences). Red blood cells were lysed with BD Pharm Lysing Buffer (BD Biosciences). Fc receptors were blocked with CD16/CD32 Fc Block antibody (BD Biosciences) followed by staining with fluorochrome-conjugated antibodies. A FACS LSR II or LSR Fortessa instrument (BD Biosciences) was used for flow cytometry acquisition. Analysis of data was done with FlowJo Software (Treestar).
10
Isolation of Pulmonary Leukocytes Protocol
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For pulmonary leukocyte isolation, lungs were cut into small pieces and then digested in RPMI medium containing 5% fetal calf serum, 250 U/ml of type IV collagenase (Invitrogen), 100 mg/ml of DNase I (Roche), and 1 mmol/l of ethylenediaminetetraacetic acid (pH 8.0), at 37 °C for 30 min. The homogenate was then filtered with a 70-mm nylon cell strainer (BD Biosciences), washed with HBSS medium, and resuspended in 40% Percoll (Amersham Pharmacia) in Dulbecco’s modified Eagle’s medium (BioWhittaker). The suspension was underlaid with 80% Percoll and centrifuged for 25 min at 1000×g. Leukocytes were collected from the interphase, washed, and enumerated on Sysmex KX-21. Fluorescent-conjugated antibodies used for analyses included CD3 (clone 500A2, 145-2C11), CD8 (clone 53–6.7), and CD4 (clone GK1.5), which were obtained from BD Biosciences Pharmingen; MHC II (clone M5/114.15.2), Gr-1 (clone RB6-8C5), B220 (clone RA3-6B2), CD11c (clone N418), F4/80 (clone BM8), CD11b (clone M1/70), and PD-L1 (12-5983-42), which were obtained from eBioscience; and CD45 (clone 30-F11), which was obtained from Invitrogen. Labeled cells were analyzed on an LSR II flow cytometer (Becton Dickinson). Data were analyzed with FCS Express software.
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