For the isolation of splenocytes, spleens were aseptically removed from recently sacrificed mice and the tissue was transferred to a tube containing 1× PBS on ice. Splenocyte suspensions were prepared by blandly squeezing the spleen between the frosted ends of the two sterile microscope slides into a 100 mm tissue culture grade Petri dish. The slides were rinsed at regular intervals with 1× PBS. The cells were formed into a single suspension using a pipette. After that, the cell suspensions were filtered through a cell strainer. Next, the cell suspensions were centrifuged at 1,500 rpm at 4°C for 5 minutes to produce pellets. For the optimal lysis of erythrocytes, the pellets were resuspended in 5 mL of red blood cell lysis buffer and incubated on ice for 5 minutes with occasional shaking. The reaction was stopped by diluting the lysis buffer with 25 mL of 1× PBS. Thereafter, cells were spun (1,500 rpm at 4°C for 5 minutes), and the supernatant was carefully removed. The pellet was then washed two times in 1× PBS and resuspended in Roswell Park Memorial Institute (RPMI)-1640 supplemented with 3% FBS and 1% antibiotic–antimycotic. Cells were then counted. The viability of the cells used in all the experiments was higher than 95%, as measured by the trypan blue exclusion method (Sigma-Aldrich, St Louis, MO, USA).
Trypan blue exclusion method
Manufactured by Merck Group
Sourced in United States, Germany
Trypan blue exclusion method is a laboratory technique used to determine the viability of cells. It involves the use of the dye trypan blue, which selectively stains dead or damaged cells blue, while live cells remain unstained. This method allows for the quantification of viable cells in a sample.
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Lab products found in correlation
Normal human dermal fibroblasts (nhdf), by PromoCell (2 mentions)
Fluoroblok cell culture inserts, by BD (1 mentions)
Dm irb inverted fluorescence microscope, by Leica (1 mentions)
Normal human dermal fibroblasts (nhdf), by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
Rpmi 1640, by Cytiva (1 mentions)
Nih 3t3, by Merck Group (1 mentions)
Thp 1, by American Type Culture Collection (1 mentions)
Mda mb 468, by Merck Group (1 mentions)
F6765, by Merck Group (1 mentions)
Hs578t, by Merck Group (1 mentions)
Mda mb 468, by American Type Culture Collection (1 mentions)
Hs578t, by American Type Culture Collection (1 mentions)
Mda mb 231, by Merck Group (1 mentions)
Nih3t3, by American Type Culture Collection (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Rpmi 1640, by GE Healthcare (1 mentions)
Trypsin, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
14 protocols using trypan blue exclusion method
1
Isolation of Murine Splenocytes
2
Lung Cancer Cell Proliferation and Migration
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Lung cancer cells were seeded in a 12-well plate at 2 × 105 cells and proliferating cells were counted after 72 and 120 h by the trypan blue exclusion method (Sigma Aldrich).
For the migration assay, we used FluoroBlok Cell Culture Inserts (BD Biosciences, San Jose, CA, USA). Briefly, 105 cells were seeded on the top chamber of the insert and RPMI plus 10% FBS was added to the bottom chamber and incubated at 37 °C and 5% CO2. After 24 h, migrated cells were fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI). Migrated cells were counted using fluorescence microscopy. Migration graphs represented the number of migrated transfected cells versus control cells. Each experiment was performed in triplicate.
For the migration assay, we used FluoroBlok Cell Culture Inserts (BD Biosciences, San Jose, CA, USA). Briefly, 105 cells were seeded on the top chamber of the insert and RPMI plus 10% FBS was added to the bottom chamber and incubated at 37 °C and 5% CO2. After 24 h, migrated cells were fixed and stained with 4′,6-diamidino-2-phenylindole (DAPI). Migrated cells were counted using fluorescence microscopy. Migration graphs represented the number of migrated transfected cells versus control cells. Each experiment was performed in triplicate.
3
Cell Viability Assay with PI and Trypan Blue
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Cell viability was measured using propidium iodide (PI, Sigma, P2667-2OTST). Cells were co-transfected with GFP in six well plates. After treatment, wells were washed with PBS once and successively incubated for 10 min with 3 µg/ml of PI in RM. Three washes with PBS were used to remove excess stain. The cells were then maintained in RM and imaged immediately using the Leica DMIRB inverted fluorescence microscope. The proportion of PI-positive cells within the transfected population was used as a measure of viability. The trypan blue exclusion method (100, 300 µM, Sigma) was also enrolled, and cells counted under the KOVA® Glasstic Slide with Counting Grids.
4
Culturing and Seeding Normal Human Dermal Fibroblasts on Hydrogels
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The primary cells, normal human dermal fibroblasts (NHDF) (Promocell, Germany), were cultured in DMEM supplemented with 10% (v/v) FCS and 1% (v/v) antibiotic-antimycotic, at 37°C, with a controlled atmosphere of 5% CO2 and 95% relative humidity. Monolayer of NHDF in their growth phase (∼90% confluence) was detached using trypsin/1 mM ethylenediaminetetraacetic (EDTA) (Life Tech., Germany) in PBS, centrifuged and resuspended in complete cell culture medium. Cells were counted using trypan blue exclusion method (Sigma-Aldrich, Germany) before seeding on hydrogels.
The prepared circular films of alginate and ADA-GEL hydrogels were placed in the wells of 24-well plates (VWR Int., Germany) and washed with DMEM. For analysis of cell adhesion, 85000 cells/film were seeded and incubated in a humidified atmosphere of 95% relative humidity and 5% CO2, at 37°C. Culture medium was changed on the next day after seeding, and then every two days.
The prepared circular films of alginate and ADA-GEL hydrogels were placed in the wells of 24-well plates (VWR Int., Germany) and washed with DMEM. For analysis of cell adhesion, 85000 cells/film were seeded and incubated in a humidified atmosphere of 95% relative humidity and 5% CO2, at 37°C. Culture medium was changed on the next day after seeding, and then every two days.
5
Isolation of Murine Splenocytes
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To isolate splenocytes, the spleen was removed aseptically from C57BL/6 mice at the endpoint treatment and was placed in a tube containing 1× PBS on ice. Splenocyte suspensions were prepared by gently pressing the spleen between the frosted ends of two sterile microscope slides into a 100 mm tissue culture grade petri dish. The slides were rinsed at regular intervals with 1× PBS. Cell suspensions were filtered by a sterile plastic strainer and then centrifuged at 1,500 rpm for 3 minutes. For optimal lysis of erythrocytes, the pellets were resuspended in 5 mL red blood cell lysis buffer and incubated on ice for 5 minutes with occasional shaking. The reaction was stopped by diluting the lysis buffer with 25 mL of 1×PBS. Thereafter, the cells were spun (1,500 rpm at 4°C for 5 minutes), and the supernatant was carefully removed. The pellet was then washed two times in 1X PBS and resuspended in Roswell Park Memorial Institute (RPMI)-1640 supplemented with 3% FBS (Hyclone Laboratories Inc.) and 1% antibiotic-antimycotic (Invitrogen Corporation). Cells were then counted. The viability of the cells used in all the experiments was higher than 95%, as measured by the trypan blue exclusion method (Sigma-Aldrich, St Louis, MO, USA).
6
Culturing Various Cell Lines
7
Cryopreserved HUCB Mononuclear Cell Preparation
8
Enzyme-Mediated Cell Transduction
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Cells were seeded in 48-well plates and incubated for 48 h to reach 80 % confluence. Then, cultures were treated with collagenase or hyaluronidase enzymes or combination of both, as indicated. Enzyme solutions were removed from cultures and washed with PBS before treatment with AAVP vectors carrying the Herpes Simplex Virus-thymidine kinase (HSVtk) gene. Ganciclovir (GCV, Sigma) was added to the cells (40 μM) at day 3 post vector transduction and renewed daily. Viable cells were monitored under microscope and cell viability was measured at day three post GCV treatment by using the trypan blue exclusion method (Sigma). When treating the M21-tk cell population stably expressing the HSVtk gene, GCV was added to cells the next day following enzymatic treatments and renewed daily for 3 days.
9
Cell Proliferation Kinetics Quantification
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Cells were initially seeded at 1 × 103 cells per cm2 in complete medium with continuous subculture for every 4 days, and the cell number was counted by the Trypan blue exclusion method (Sigma‐Aldrich) for a total of five passages. Cumulative population doublings (CPDs) were calculated by using the formula: CPD = [log10 (NH) − log10(N1)]/log102, where NH is the harvested cell number and N1 is the plated cell number. The generation time was calculated as: [log102 × Δt]/[log10(NH) − log10(N1)], where Δt is the time between passages 11 .
10
Perfusion Culture of 14F7htb58 Cells
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Example 2
14F7htb58 cells adapted to growth in protein-free PFHMII cell culture medium were inoculated in a 5 L fermenter at a concentration of 0.4-0.5×106 cell/ml. Fermenter operation parameters were: pH 6.9-7.0; 105 RPM; 40% dissolved oxygen; 36.5-37.0° C.; work volume 3.5 L. Cell viability and cell concentration were monitored daily by Trypan Blue exclusion method (Sigma) using a Neubauer chamber. Fermenter operation in perfusion mode was performed after cell concentration reached the value of 2.5×106 cell/ml. A hollow fiber cartridge was employed for the perfusion mode and a dilution rate of 0.3-0.7 VVD was used.
During the fermentation process cell viability kept over 90% up to 384 hours, and then it was reduced to 80%. Antibody concentration was in the range of 30-40 mg/L. The maximal cell concentration reached was 9×106 cell/ml when a dilution rate of 0.7 VVD was used (
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