HUVECs or LUAD cells were incubated with PBS or indicated exosomes (10 μg) for 24 h. Cells (1 × 105) were then harvested and cultured in serum-free medium in 24-well transwell upper chambers (Corning) without Matrigel coating. The lower chambers were filled with complete medium. After 24 h, the migrated HUVECs were fixed with methanol and stained with 0.1% crystal violet solution (Sigma-Aldrich). The stained cells from five random visual fields were then counted using a light microscope.
24 well transwell upper chambers
Manufactured by Corning
Sourced in United States
The 24-well transwell upper chambers are laboratory equipment designed for cell culture and migration studies. They feature a permeable membrane insert that can be placed into a 24-well plate, allowing for the separation of cell populations and the study of cellular interactions and migration.
Automatically generated - may contain errors
Lab products found in correlation
Ordinary optics microscope, by Olympus (2 mentions)
Matrigel, by Corning (2 mentions)
Crystal violet solution, by Merck Group (1 mentions)
Upper transwell chamber, by Corning (1 mentions)
Matrigel, by BD (1 mentions)
Phorbol myristate acetate (pma), by Sangon (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
7 protocols using 24 well transwell upper chambers
1
Exosome-Induced Cell Migration Assay
2
Cell Invasion Assay Protocol
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Cells (0.1–1 × 106) were resuspended in 200 μL serum-free medium and seeded in the 24-well transwell upper chambers (Corning, Corning, NY, USA). A total of 500 μL RPMI 1640 medium containing 10% FBS was added into the lower chambers. After culturing for 12–48 h, cells on the membrane were fixed with formalin, stained with H&E, and then calculated and photographed using the ordinary optics microscope (Olympus). Invasion assays were performed according to the same procedures with the diluted Matrigel (Corning) (RPMI 1640: Matrigel = 5:1) spread on the inside bottom of the transwell upper chambers.
3
Cell Migration Assay Protocol
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200 μL serum-free medium containing 2 × 104 transfected cells were seeded into 24 well transwell upper chambers (Corning, USA), respectively. 600 μL of complete medium with 10% FBS was added to the lower chamber. After incubation for 24 h, the non migrated cells on the surface of the upper chamber were erased, then the upper chamber was fixed with 4% paraformaldehyde for 30 min at room temperature and stained with 1% crystal violet for 5 min. The staining results were observed under a microscope and five fields were randomly selected to count the cells. All assays were independently performed in triplicate.
4
Transwell Invasion Assay Protocol
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Cells (0.1-1×10 6 ) were resuspended in 200µL serum-free medium and seeded in the 24-well transwell upper chambers (Corning, NY, USA). 500µL RPMI-1640 medium containing 10% FBS was added into the lower chambers. After culturing for 12-48h, cells on the membrane were xed with formalin, stained with hematoxylin and then calculated and photographed using the ordinary optics microscope (Olympus). Invasion assays were performed according to the same procedures with the diluted Matrigel (Corning) (RPMI-1640: Matrigel = 5:1) spread on the inside bottom of the transwell upper chambers.
5
Wound Healing and Invasion Assays
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For the wound-healing migration assay, SUN1076 or FaDu cells were cultured for 24 h on six-well plates with DMEM containing 10% FBS and grown to 100% confluency. A scratch was made in each well using a pipette tip. Cells were washed twice with PBS and cultured in serum-free medium containing 25% medium without FBS, CAFCM, or NFCM for 24–72 h. Images were captured at 0, 24, 48, and 72 h. Finally, ImageJ (National Institutes of Health, Bethesda, MD, USA) was used to measure migration distances.
For the transwell invasion assay, 1 × 105 SUN1076 or FaDu cells were seeded on upper 24-well transwell chambers (Corning, USA, 3422) coated with 80 μL of Matrigel (BD Biosciences, USA; 356234) in serum-free DMEM. Medium containing 20% FBS was added to the lower chambers for 48 h to induce chemotaxis. Cells that migrated through the 8-μm pores were fixed with methanol and stained with 0.1% crystal violet. Stained cells were visualized using a microscope and those in five random fields were counted.
For the transwell invasion assay, 1 × 105 SUN1076 or FaDu cells were seeded on upper 24-well transwell chambers (Corning, USA, 3422) coated with 80 μL of Matrigel (BD Biosciences, USA; 356234) in serum-free DMEM. Medium containing 20% FBS was added to the lower chambers for 48 h to induce chemotaxis. Cells that migrated through the 8-μm pores were fixed with methanol and stained with 0.1% crystal violet. Stained cells were visualized using a microscope and those in five random fields were counted.
6
Evaluating Cell Proliferation and Migration
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CCK8 assay (MCE, American) was used to analyze cell proliferation. 1 × 103 cells were cultured in 96-well plates for 24 h, 48 h and 72 h. Then, after incubation, 10 μL of CCK8 reagent was added into every well of 96-well plates and incubated for 2 h. After incubation of CCK8 reagent, we measured the OD value at 450 nm.
For the wound healing assay, the cells were plated in six-well plates, and cultured until 90% confluent. The confluent monolayer was wounded with a 200-μL pipette tip, and the unattached cells were removed. The scratches were observed at 0 h and 24 h after incubation of the monolayers in the FBS-free medium.
For migration assay, 1 × 104 bladder cancer cells were seeded on the upper 24-well transwell chambers (Corning) and culture for 24 h. After incubation, the cells move to the bottom of the 24-well chamber, followed by fixing with 4% formaldehyde and dyeing with crystal violet reagent.
For the wound healing assay, the cells were plated in six-well plates, and cultured until 90% confluent. The confluent monolayer was wounded with a 200-μL pipette tip, and the unattached cells were removed. The scratches were observed at 0 h and 24 h after incubation of the monolayers in the FBS-free medium.
For migration assay, 1 × 104 bladder cancer cells were seeded on the upper 24-well transwell chambers (Corning) and culture for 24 h. After incubation, the cells move to the bottom of the 24-well chamber, followed by fixing with 4% formaldehyde and dyeing with crystal violet reagent.
7
Coculture System: LUAD Cell Progression
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Human LUAD cells (A549 and H1975), normal lung epithelial cells (BEAS‐2B) monocytes (THP‐1), and embryonic kidney cells (293 T) (Procell) were cultured at 37°C with 5% CO2. A549, H1975, BEAS‐2B and 293T cells were cultured in Ham's F‐12 K medium, RPMI‐1640 and DMEM (Gibco) plus 10% FBS and 1% streptomycin/penicillin, respectively, and THP‐1 cells were grown in RPMI‐1640 (Gibco) plus 0.05 mM β‐mercaptoethanol, 10% FBS and 1% streptomycin/penicillin. THP‐1 cells were treated with 100 ng/mL PMA (Sangon) for 24 h to induce M0 macrophages. To explore the effect of M2 macrophage polarization on LUAD cell progression, THP‐1 + PMA transfected with si‐circ_0001715/si‐NC or incubated with exosomes were seeded in the upper 24‐well transwell chambers (Corning Inc.) to form the conditioned medium, and LUAD cells were seeded in the lower chambers to build a coculture system. After 24 h, LUAD cells were collected for analysis.
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