Flow cytometry was used to quantify the percentage of cells in organs that were Cy7 positive or td-Tomato positive following treatment. Single-cell suspensions were obtained as described previously [49 ,50 ] for further staining and flow cytometry analysis. Freshly dissected the heart, the liver, lungs, kidneys, the GI tract, and the brain were minced and digested with 500 μL of 1 mg/mL collagenase type I (Gibco) by incubating at 37 °C, 5% CO2 for 20 min in 1.5 ml Eppendorf tubes. Cell suspension was collected, neutralized with a medium containing 10% fetal bovine serum (FBS), and placed on ice. Fresh collagenase solution was added to the remaining tissue and incubated for an additional 20 min. Cell suspensions were filtered through 70um Nylon cell strainer (Falcon, cat. 352,350) to create a single-cell suspension. The Attune NxT Flow Cytometer (Thermo Fisher Scientific) was used for performing flow cytometry, and FlowJo software (FlowJo LLC) was used for data analyses. All antibodies were obtained from BD Biosciences. Cells were stained with BV421-CD56 (cat.748,094), APC-CD31 (551,262), FITC-CD90.2 (553,003), BV421-CD45 (563,890), BV650 CD324 (752,472), FITC-CD71 (561,936), APC-Ter119 (557,909) from BD Biosciences. BDTM anti-Rat Ig, κ CompBeads (552,844) were used to generate compensation controls.
70um nylon cell strainer
Manufactured by BD
Sourced in United States
The 70um Nylon cell strainer is a laboratory equipment designed to filter and separate cells from a cell suspension. It features a nylon mesh with 70 micrometer pore size to capture and retain cells while allowing smaller particles and debris to pass through.
Automatically generated - may contain errors
Lab products found in correlation
Cd31 apc, by BD (1 mentions)
Ter119 apc, by BD (1 mentions)
Cd56 bv421, by BD (1 mentions)
Cd45 bv421, by BD (1 mentions)
Cd71 fitc, by BD (1 mentions)
Attune nxt flow cytometer, by Thermo Fisher Scientific (1 mentions)
Collagenase type 1, by Thermo Fisher Scientific (1 mentions)
Blood cell lysis buffer, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
2 mercaptoethanol, by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Ack lysis buffer, by Thermo Fisher Scientific (1 mentions)
3 protocols using 70um nylon cell strainer
1
Multiparametric Flow Cytometry Analysis of Organ Cells
2
Splenocyte Isolation and Activation
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Spleens were harvested in ice-cold RPMI-1640 medium (Gibco, Stockholm, Sweden) and stored on ice until processing within hours. Spleens were pressed against a 70 um nylon cell strainer (Falcon, New York, NY, USA) to obtain a single cell suspension of splenocytes, followed by a washing step with RPMI. The pellets were resuspended and mixed in blood cell lysis buffer (Sigma-Aldrich, St Louis, MO, USA) to remove erythrocytes. Cells were counted and suspended in complete RPMI containing 1 mmol/L sodium pyruvate, 10 mmol/L Hepes, 50 U penicillin, 50 µg/mL streptomycin, 0.05 mmol/L 2-mercaptoethanol, 2 mmol/L L-glutamine and 10% heat inactivated fetal bovine serum (FBS) all purchased from Gibco (Stockholm, Sweden). One million splenocytes were cultured in microtiter 48-well plates and stimulated for 18 h with anti-CD3/CD28 beads. Cells were spun down at 1000 g for 5 min at 4 °C and supernatants were stored at −80 °C until further analysis.
3
Murine Splenic Immune Cell Isolation
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Two weeks following the final immunization, mice were euthanized and spleens removed. Single cell splenocyte preparations were obtained by passage through a 70 um nylon cell strainer (BD Falcon). Erythrocytes were depleted with ACK lysis buffer (Gibco) and splenocytes were washed thoroughly with R10 media (RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 50 µM 2-mercaptomethanol, 100 U/ml of penicillin, 100 µg/ml streptomycin, and 10 mM HEPES).
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