T-cell chemotaxis was assayed by using 24-well Transwell chambers with 5 μm pores (Corning). A total of 2 × 105 cells in 100 μL chemotaxis buffer (RPMI 1640 with 10% FBS) were placed in the upper chambers. CCL2, diluted in 600 μL chemotaxis buffer was placed in the lower wells, as well as different cell culture supernatant, and the chambers were then incubated for 3 h in the incubator at 37 °C. Migrated cells located in the bottom wells were collected, washed once with PBS, and counted by FACS using CountBright™ Absolute Counting Beads (BD bioscience).
Countbright absolute counting beads
Manufactured by BD
CountBright Absolute Counting Beads are a set of fluorescent beads used for quantifying cell counts and particle concentrations in flow cytometry and other applications. The beads are of a known concentration and can be used to determine the absolute number of target cells or particles in a sample.
Automatically generated - may contain errors
Lab products found in correlation
Accuri, by BD (2 mentions)
Transwell chamber, by Corning (1 mentions)
Gentlemacs tube, by Miltenyi Biotec (1 mentions)
Phase flow alexa fluor 647 brdu kit, by BioLegend (1 mentions)
Liberase, by Roche (1 mentions)
Anti cd3 cd28 beads, by Thermo Fisher Scientific (1 mentions)
Cytokine bead array th1 2 17 kit, by BD (1 mentions)
6 protocols using countbright absolute counting beads
1
Transwell Assay for T-cell Chemotaxis
2
In Vivo Cell Labeling and Tissue Dissociation
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Mice were injected i.v. with 2.5 µg of Alexa Fluor 488– or Alexa Fluor 647–conjugated anti-CD45.2 (clone 104) or anti-Thy1.2 (clone 30-H12) antibody. After 5 min, lung lobes were collected and mechanically disrupted using gentleMACS tubes (Miltenyi Biotec) with a spleen 4.1 dissociation protocol. Lung tissue was incubated in Liberase TM (Roche) solution (20 µg/ml in DMEM) at 37°C for 1 h before dissociation with gentleMACS. LN cells were minced and incubated at 37°C in Liberase TM solution and mechanically dissociated by pipetting up and down. The spleen was minced and incubated at 37°C for 30 min in Liberase TM solution before being ground through a 70-µm filter. Cells were stained for 30 min at room temperature in PBS containing 2% FBS using the antibodies listed in Table S1 . The CD8 tetramer was specific for influenza A NP linear epitope ASNENMETM (MBL International). For BrdU detection, cells were fixed and permeabilized using the Phase-Flow Alexa Fluor 647 BrdU kit (BioLegend). Cell numbers were determined using CountBright Absolute Counting Beads (BD Biosciences). All samples were acquired on a BD Fortessa, FACSCanto, or LSRII flow cytometer. Data were analyzed using FlowJo version 10 software (Tree Star).
3
Transwell Migration Assay for CXCL12
4
Regulatory T cell Differentiation
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Sorted Th cells from healthy donors were activated with anti-CD3/CD28 beads (1:5 ratio)(Life Technologies) in XVIVO-15 media for 5 days a 37°C. Then, prostaglandin E2 (10 uM), 1,25(OH)VitD (10nM) and recombinants chemokines CCL1 and CCL18 (0.5ug/mL) were added to 1x105 Th in XVIVO-15 serum-free medium for 72h a 37°C. After the incubation, the supernatants were stored for further cytokine production measurement using the Cytokine Bead Array Th1/2/17 Kit (BD) and live cells were counted (CountBright Absolute Counting Beads), stained with anti-CCR8 and analyzed by flow cytometry.
5
Hox Cell Interventions and Proliferation
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Hox cells were cultured with the following interventions: electroporation with a GFP targeting sgRNA, etoposide, or AMG232. Cells were counted each day by flow cytometry (BD Accuri) with CountBright Absolute Counting Beads.
6
Hox Cell Response to Interferon Beta
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Hox cells were cultured with or without mouse Interferon Beta and the Jak1 inhibitor Solcitinib for 7 days. Cells were counted on day 7 by flow cytometry (BD Accuri) using CountBright Absolute Counting Beads.
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