To obtain splenocytes, spleens were aseptically dissected from 8-week-old Balb/c mice. The tissues were teased apart gently with forceps and then forced through a 70-μm cell strainer (SPL Life Sciences, Pocheon-si, Gyeonggi-do, S. Korea). The resulting cell suspension was washed three times in RPMI-1640 (Invitrogen, Carlsbad, CA, USA) by centrifugation (1,000 g, 5 min, 4°C) and treated with red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). The isolated splenocytes were maintained in RPMI-1640-based culture medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (all from Invitrogen) in a culture incubator (37°C, 5% CO2, humidified).
70 μm cell strainer
Manufactured by SPL Life Sciences
Sourced in Cameroon
The 70-μm cell strainer is designed for the filtration and separation of cells and tissue samples. It features a nylon mesh with a pore size of 70 micrometers, allowing for the removal of larger cell aggregates and debris while retaining the desired cell population.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Rpmi 1640, by Thermo Fisher Scientific (5 mentions)
Red blood cell lysis buffer, by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Lsrfortessa flow cytometer, by BD (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (2 mentions)
Facscanto 2, by BD (2 mentions)
Liberase tl, by Merck Group (2 mentions)
Red blood cell lysing buffer, by Merck Group (1 mentions)
Raw 264.7 macrophage cells, by Korean Cell Line Bank (1 mentions)
Ammonium chloride potassium ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Vimentin, by Abcam (1 mentions)
Cytokeratin 5, by Leica (1 mentions)
18 protocols using 70 μm cell strainer
1
Isolation and Culture of Murine Splenocytes
2
Splenic Index Calculation and Splenocyte Isolation
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The spleens obtained from ICR and BALB/c mice were washed with sterilized saline and weighed, and the following index was calculated: spleen index (mg/g) = spleen weight (mg)/B.W. (g). Spleens were stored in cold RPMI 1640 before splenocyte extraction. The spleens were gently pressed through a syringe and filtered through a 70-μm cell strainer (SPL Life Sciences, Korea). Red blood cells were removed by treatment with red blood cell lysis buffer and washed with RPMI 1640 medium.
3
Culturing RAW 264.7 Macrophages and Rat Splenocytes
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RAW 264.7 macrophage cells were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea) and incubated in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, Gaithersburg, MD, USA) and 1% penicillin/streptomycin (Invitrogen) at 37°C and 5% CO2. A single-cell suspension of splenocytes was prepared using tweezers and a 70-μm cell strainer (SPL Life Sciences, Pocheon, Gyeonggi, Korea) after aseptic excision of the spleens of Wistar rats. Cells were washed in Roswell Park Memorial Institute (RPMI)-1640 (Invitrogen) by centrifugation three times (80 × g for 3 min at 4°C). Red blood cell lysing buffer (Sigma-Aldrich) was added for 3 min to remove erythrocytes and then used for the experiment. Isolated splenocytes were cultured in RPMI-1640 medium containing 10% FBS (Gibco BRL) and 1% penicillin and streptomycin (Invitrogen) at 37°C in a humidified incubator with 5% CO2.
4
HDM-Induced Allergic Splenocyte Response
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In the in vitro experiments, mice were sensitized with HDM/alum on days 0 and 7, and sacrificed on day 14. Spleens were aseptically recovered. Single-cell suspensions were prepared by gently teasing apart the tissue in RPMI1640 medium supplemented with10% heat-inactivated FBS (Thermo Fisher Scientific), 2 mM L-glutamine (Thermo Fisher Scientific), 10 mM HEPES (Thermo Fisher Scientific), 50 μM 2-mercaptoethanol (Sigma-Aldrich), 100 U/mL penicillin–streptomycin (Thermo Fisher Scientific), and then passed through a 70-μm cell strainer (SPL Life Sciences). Red blood cells were lysed using ammonium–chloride–potassium (ACK) lysis buffer (Thermo Fisher Scientific, Waltham, MA, United States). Single-cell suspensions from spleen (splenocytes) were cultured at 1 × 106 cells/well in U-bottom 96-well plates in the presence of HDM (35 μg/mL) ± AMDK19-HK for 3 days. In the ex vitro experiments, mice were sensitized with HDM/alum on days 0 and 7, and AMDK19-HK was administered from 1 week before the first sensitization until the 1 day before the sacrifice. On day 14 splenocytes were obtained and restimulated with or without HDM for 3 days. Cytokine production was measured in supernatants by using ELISAs.
5
Isolation and Characterization of Cancer-Associated Fibroblasts
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CAFs were isolated as previously described [46 (link)]. Briefly, IDC tissues were minced and then digested overnight in a collagenase cocktail (ISU ABXIS; Seoul, South Korea). Digested tissues were filtered through a 70-μm cell strainer (SPL Life Science; Pocheon-si, South Korea). Cells were separated using Ficoll gradients, washed with phosphate-buffered saline (PBS), resuspended in Dulbecco's Modified Eagle medium (DMEM)/F12 medium containing 20% (v/v) fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Gibco BRL; Grand Island, NY, USA), and cultured at 37°C in a humidified atmosphere of 5% CO2. The fibrotic characteristics of the isolated cells were confirmed by microscopic examination of morphology and immunofluorescence analysis using antibodies against vimentin (Abcam; Cambridge, UK), cytokeratin (Dako; Glostrup, Denmark), and cytokeratin 5 (Novocastra; Newcastle upon Tyne, UK).
6
BM Cell Isolation and Flow Cytometry
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BM cells were isolated from femur and tibia and dispersed into single-cell suspensions by passing through a 70-μm cell strainer (SPL, Korea). Erythrocytes were removed by ACK lysis. Cells were blocked with anti-CD16/32 and then stained for surface molecules. DAPI (4,6-diamidino-2-phenylindole; Roche) was used for dead cell exclusion. Live cells were counted with counting beads (Invitrogen). For intracellular staining, a Live/Dead Fixable Violet Dead-Cell Stain kit (Invitrogen) was used for dead cell exclusion. Cells were fixed with 3.7% formaldehyde and quenched with 10 mM glycine/phosphate-buffered saline (PBS). Fixed cells were permeabilized with 0.5% saponin/PBS and stained for intracellular molecules. Data were acquired on an LSRFortessa flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star).
7
Isolation and Culture of PDLSCs
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This study was approved and supervised by the Institute of Review Board of Chungbuk National University (CBNU-201608-BMBRETC-322-01). Only healthy adults (18 to 30 years old) who provided informed consent for participation were enrolled in this study. Plaque on the teeth was removed with a curette, and the teeth were disinfected with chlorhexidine solution and washed with saline twice. Thenon-decayed third molars were then extracted by a dentist and stored in Dulbecco’s PBS with 1% antimycotic/antibiotic solution (Gibco, Carlsbad, CA, USA). Periodontal ligaments were separated gently from the mid-third of the root, minced into tiny pieces and immersed in a mixture of 1:1 type I collagenase (Worthington Biochem, Freehold, NJ, USA)/dispase II solution. Cells were incubated at 37 ℃ for 60–90 minutes with gentle shaking and isolated to a single-cell suspension using a 70-
μm cell strainer (SPL Life Science, Seoul, Republic of Korea). PDLSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% FBS (Gibco) and 1% nonessential amino acids (Gibco), hereafter referred to as growth medium, under humidified conditions of 5% CO2 in air at 37 ℃.
μm cell strainer (SPL Life Science, Seoul, Republic of Korea). PDLSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) containing 10% FBS (Gibco) and 1% nonessential amino acids (Gibco), hereafter referred to as growth medium, under humidified conditions of 5% CO2 in air at 37 ℃.
8
Isolation of Rat Splenocytes
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The spleen of an 8-week-old SD rat was aseptically dissected to obtain splenocytes. The spleen was gently pressed with forceps and then forced through a 70-μm cell strainer (SPL Life Sciences, Pocheon-si, Gyeonggi-do, Korea). The cells were collected and washed three times in Roswell Park Memorial Institute (RPMI)-1640 (Invitrogen, Carlsbad, CA, USA) by centrifugation (80 × g, 3 min, 4°C). Next, the cells were treated with red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). Isolated splenocytes were maintained in RPMI-1640 containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin, streptomycin) (Invitrogen) in a 5% CO2 incubator.
9
Isolation and Culture of Rat Splenocytes
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Cells were cultured as previously described [20 (link)]. Briefly, the spleen of an 8-week-old SD rat was aseptically dissected to obtain splenocytes. The spleen was gently pressed with forceps and then forced through a 70-μm cell strainer (SPL Life Sciences, Pocheon-si, Gyeonggi-do, Korea). Cells were treated with red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO, U.S.A.). Isolated splenocytes were incubated in RPMI-1640 containing 10% fetal bovine serum (FBS) and 1% antibiotics (penicillin, streptomycin) (Invitrogen) in a 5% CO2 incubator.
10
Isolation of Colonic Cells from Mice
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The large intestines of mice were removed and washed in ice-cold PBS (Invitrogen), and the fat was removed. The intestines were opened longitudinally, washed in PBS, and cut into 1-cm sections. Whole colonic segments were prepared by incubation with 5 ml of an enzyme mixture containing 0.5 M EDTA (Duchefa) and 1 M DTT (Goldbio) in Hank's balanced salt solution without calcium or magnesium (Welgene) for 30 min at 37 °C with gentle mixing. Cell suspensions were prepared by filtration using a 100-μm cell strainer (SPL Life Sciences), 5 ml of PBS was added, the filtration step was repeated, and the remaining tissue was cut into 0.5-mm sections using scissors. The pieces were digested with 5 ml of enzyme mixture containing collagenase D (Roche), DNase I (Sigma Aldrich), and Dispase II (Sigma Aldrich) in Hank's balanced salt solution with calcium and magnesium, using a gentleMACS C tube attached to a gentleMACS Dissociator (Miltenyi Biotec). The mixture was incubated for 30 min at 37 °C with automated rotation and filtered using a 70-μm cell strainer (SPL Life Sciences), and the digestion step (5 ml enzyme mixture) was repeated. The sample was centrifuged at 13,000 rpm for 10 min at 4 °C, and the cell pellets were collected.
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