Proliferating MDA-MB-231 cells were trypsinized and counted using Cellometer Auto T4 Cell Counter. A cell suspension of 100,000 cells/mL in growth medium was prepared and 100 μL of the suspension was loaded into each BD Matrigel 24-well 8.0 μm PET Membrane Invasion Chamber (#354483). Matrigel coated plates, and control insert plates had 500 μL NIH3T3-conditioned medium loaded in the bottom as the chemoattractant. Plates and chemoattractant medium were incubated at 37 °C for 3–4 h prior to loading MDA-MB-231 cells. Cells were incubated for 16 h at 37°C in 5% CO2 and then fixed and stained using the Fisher HealthCare PROTOCOL Hema 3 Manual Staining System (#22-122-911) according to the manufacturer’s instructions. Cotton swabs were used to eliminate cells which did not migrate/invade as well as Matrigel. Cells were counted using an inverted light microscope. To control for proliferation effects, rates of cellular invasion through Matrigel were normalized by rates of cellular migration through control plastic-only insert wells.
Protocol hema 3 manual staining system
Manufactured by Thermo Fisher Scientific
The PROTOCOL Hema 3 Manual Staining System is a laboratory instrument designed for the manual staining of hematology samples. It provides a controlled and consistent method for staining blood smears to facilitate the microscopic examination and analysis of cellular components.
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4 protocols using protocol hema 3 manual staining system
1
Invasion Assay for MDA-MB-231 Breast Cancer Cells
2
Evaluating Cell Growth and Invasion
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Cell growth was assessed
with clonogenic assays. Briefly, A2780CP20 cells (2 × 104 cells/mL) were seeded into six-well plates, and 24 h later,
cells were transfected with siRNAs encapsulated on the HiPerfect transfection
reagent, regular liposomes, or βCD-liposomes. The next day,
cells were collected, and 1000 transfected cells were seeded in 10
cm Petri dishes. Colonies formed after seven days were stained with
0.5% crystal violet in methanol. Colonies of at least 50 cells were
quantified under a light microscope (CKX41; Olympus) at 10× magnification
in five random fields. Percentages of clonogenicity were calculated
relative to the NC-siRNA. To assess cell invasion, cells (2 ×
104 cells/mL) were seeded in 10 cm Petri dishes. Twenty-four
hours later, cells were transfected with siRNAs encapsulated on the
HiPerfect transfection reagent, regular liposomes, or βCD-liposomes.
The next day, 70,000 cells were seeded into matrigel-coated transwells.
Forty-eight hours later, cells were fixed and stained using the Fisher
HealthCare PROTOCOL Hema 3 Manual Staining System. The invading cells
were counted at 20X on an Olympus 1X71 microscope equipped with a
digital camera (Olympus DP26). Percentages of invaded cells were calculated,
taking the untransfected cell values as 100% of cell invasion.
with clonogenic assays. Briefly, A2780CP20 cells (2 × 104 cells/mL) were seeded into six-well plates, and 24 h later,
cells were transfected with siRNAs encapsulated on the HiPerfect transfection
reagent, regular liposomes, or βCD-liposomes. The next day,
cells were collected, and 1000 transfected cells were seeded in 10
cm Petri dishes. Colonies formed after seven days were stained with
0.5% crystal violet in methanol. Colonies of at least 50 cells were
quantified under a light microscope (CKX41; Olympus) at 10× magnification
in five random fields. Percentages of clonogenicity were calculated
relative to the NC-siRNA. To assess cell invasion, cells (2 ×
104 cells/mL) were seeded in 10 cm Petri dishes. Twenty-four
hours later, cells were transfected with siRNAs encapsulated on the
HiPerfect transfection reagent, regular liposomes, or βCD-liposomes.
The next day, 70,000 cells were seeded into matrigel-coated transwells.
Forty-eight hours later, cells were fixed and stained using the Fisher
HealthCare PROTOCOL Hema 3 Manual Staining System. The invading cells
were counted at 20X on an Olympus 1X71 microscope equipped with a
digital camera (Olympus DP26). Percentages of invaded cells were calculated,
taking the untransfected cell values as 100% of cell invasion.
3
Quantifying Cell Proliferation and Invasion
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Cell proliferation was assessed with clonogenic assays. Cells (3.0 × 104 cells/mL) were seeded into 6-well plates, and 24 h later, the cells were transfected with OMIs (100 nM final concentration) as described above. The next day, the transfected cells (1000) were seeded in 10 cm Petri dishes. After seven days, the colonies that had formed were stained with 0.5% crystal violet in methanol. Colonies of at least 50 cells were quantified under a light microscope (CKX41; Olympus) at 10× magnification in five random fields. The percentages of clonogenicity were calculated relative to the control. To assess cell invasion, cells (2 × 104 cells/mL) were seeded in 10 cm Petri dishes. Twenty-four hours later, the cells were transfected with OMIs (50 nM final concentration). The next day, 70,000 cells were seeded into Matrigel-coated transwells. Forty-eight hours later, the cells were fixed and stained using the Fisher HealthCare™ PROTOCOL™ Hema 3™ Manual Staining System. The invading cells were counted at 20× on an Olympus 1 × 71 microscope equipped with a digital camera (Olympus DP26). The percentages of invaded cells were calculated, taking the untransfected cell values as 100% of the cell invasion.
4
Hema 3 Manual Staining of Transformed Cells
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0.1 to 1 × 105 cells treated with AS-99 , AS-nc or 0.25% DMSO were collected at day 7 for staining. Cytospins were prepared and stained using the PROTOCOL™ Hema 3™ Manual Staining System (22-122911, Fisher Scientific) as described before45 (link). For WT or ΔSET Ash1l murine bone marrow cells transformed with various oncogenes, cells were collected after fourth round of colony assay, washed twice with PBS, and counted. 0.5 to 1 × 105 cells in 100 µL PBS were used for cytospins and staining by applying the same procedure as described before45 (link).
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