The collected whole blood (1.5 mL) samples were diluted in 2.5 mL of Hank’s balanced salt solution (HBSS) and carefully layered into 15 mL SepMateTM tubes (StemCell Technologies, Vancouver, BC, Canada) containing 3.5 mL Ficoll Plaque Premium (GE Healthcare, Chicago, IL, USA). The tubes were centrifuged for 15 min (min) at 1200× g, 20 °C. The upper layer consisting of PBMCs was decanted into a 15 mL falcon tube containing 7 mL of HBSS. Following mixing, the tubes were centrifuged at 400× g (18 °C) for 15 min. Following centrifugation, the supernatant was discarded, and the pellet was resuspended in 7 mL of HBSS. The resuspended cells were centrifuged again under the same conditions. Then, the supernatant was discarded, and the pellet was resuspended in Roswell Park Memorial Institute medium (RPMI; Gibco, Carlsbad, CA, USA) containing 1% l-glutamine, 1% of antibiotic (100 U/mL penicillin and 100 µg/mL streptomycin) and 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA). The PBMCs in each sample were counted using LUNATM Cell Counting Slides (Logos Biosystems, Annandale, VA, USA) with the LunaTM Automated Cell Counter (Logos Biosystems, Annandale, VA, USA).
Luna cell counting slide
Manufactured by Logos Biosystems
Sourced in United States, Cameroon, Germany
The Luna cell counting slide is a laboratory equipment designed for cell counting. It provides a standardized and consistent platform for accurately determining the concentration of cells in a given sample.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Luna fl automated fluorescence cell counter, by Logos Biosystems (2 mentions)
Luna automated cell counter, by Logos Biosystems (1 mentions)
Sepmate tube, by STEMCELL (1 mentions)
Ficoll plaque premium, by GE Healthcare (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
Luna iitm automated cell counter, by Logos Biosystems (1 mentions)
Luna fl automated cell counter, by Logos Biosystems (1 mentions)
Acridine orange propidium iodide stain, by Logos Biosystems (1 mentions)
Luna fl dual fluorescence cell counter, by Logos Biosystems (1 mentions)
Trypan blue, by Merck Group (1 mentions)
Cell proliferation kit 2 xtt, by Merck Group (1 mentions)
Luna 2, by Logos Biosystems (1 mentions)
Accutase, by Merck Group (1 mentions)
Synergy ht, by Agilent Technologies (1 mentions)
Dm il led, by Leica (1 mentions)
9 protocols using luna cell counting slide
1
Isolation and Enumeration of PBMCs
2
Evaluation of Cell Growth in Conditioned Media
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In Huh7 conditioned medium. 1.5⨯10 5 MV3 cells/well were seeded into 6-well plates and cultured in RPMI 1640 medium conditioned by either Huh7 or MV3 cells over a period of 120 h. The conditioned medium accounted for 33% of the total medium amount. After 48 h, three wells per condition were trypsinized and pooled for cell counting. To discriminate between live and dead cells, 10 µl of each sample were mixed with 10 µl trypan blue solution before being counted by an automated cell counter (LUNA-II TM automated cell counter & LUNA TM cell counting slides, Logos Biosystems, Donga-gu-Anyang-si, South Korea). Each sample was measured in triplicate.
In serum-containing medium. 3⨯10 4 MV3 cells/well were seeded into 24-well plates and exposed to drug-containing media. After 24 h, ATII was renewed and after another 24 h, proliferation was determined. Four wells per condition were trypsinized and pooled for counting in duplicate according to the trypan blue procedure.
In serum-free medium. 24 h serum-starved MV3 cells were seeded into a 24-well plate at a density of 6⨯10 4 cells/well and exposed to the selected drugs for 96 h. ATII was renewed every 24 h and proliferation analyzed as described for serum-containing medium.
In serum-containing medium. 3⨯10 4 MV3 cells/well were seeded into 24-well plates and exposed to drug-containing media. After 24 h, ATII was renewed and after another 24 h, proliferation was determined. Four wells per condition were trypsinized and pooled for counting in duplicate according to the trypan blue procedure.
In serum-free medium. 24 h serum-starved MV3 cells were seeded into a 24-well plate at a density of 6⨯10 4 cells/well and exposed to the selected drugs for 96 h. ATII was renewed every 24 h and proliferation analyzed as described for serum-containing medium.
3
Cell Culture and Counting of SH-SY5Y Neuroblastoma
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The human neuroblastoma cell line (SH‐SY5Y) was used in this investigation. SH‐SY5Y cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Cat. No. 11995065; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific) and 1% penicillin–streptomycin (Thermo Fisher Scientific). Cells were incubated in a Falcon 25‐cm2 Rectangular Canted Neck Cell Culture Flask with Vented Cap (Cat. No. 353108; Corning, Corning, NY, USA) or a Falcon four‐well Culture Slide (Cat. No. 354114; Corning) and maintained at 37°C in a humidified incubator under 5% CO2 atmosphere. Cell counting was verified by the trypan blue dye exclusion method using a LUNA‐FL automated cell counter (Logos Biosystems, Gyeonggi‐do, South Korea) and dedicated using LUNA Cell Counting Slides (Logos Biosystems). SH‐SY5Y cells were divided into different groups with or without several treatments as mentioned later.
4
Radiolabeling of PLGA Nanoparticles for moDC Uptake
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NPs were 111In-labeled as described above.
This experiment was performed in triplicate. MoDC cells were incubated
with [111In]In-PLGA-NH2 NPs (5.52 MBq/mg) in
RPMI-1640 at culture conditions for 2 h. In parallel, moDC cells were
labeled with [111In]In-oxine (Curium Netherlands B.V.,
The Netherlands) method for similar specific activity and incubated
at RT on a shaker for 15–20 min. After labeling, cells were
washed with PBS and resuspended in RPMI-1640 medium and incubated
at culture conditions for 1, 2, 4, 6, 24, and 48 h. To determine the
retention of the NPs by the moDC cells, the amount cell-associated
radioactivity was measured before and after one wash step at every
time point. Radiolabel retention was calculated as the fraction still
cell associated after washing. Cells were stained with trypan blue
and counted with Luna Cell Counting Slides (Logos biosystems, South
Korea). The measured activity per sample and the number of cells counted
were used to calculate the specific activity per cell or million cells.
This experiment was performed in triplicate. MoDC cells were incubated
with [111In]In-PLGA-NH2 NPs (5.52 MBq/mg) in
RPMI-1640 at culture conditions for 2 h. In parallel, moDC cells were
labeled with [111In]In-oxine (Curium Netherlands B.V.,
The Netherlands) method for similar specific activity and incubated
at RT on a shaker for 15–20 min. After labeling, cells were
washed with PBS and resuspended in RPMI-1640 medium and incubated
at culture conditions for 1, 2, 4, 6, 24, and 48 h. To determine the
retention of the NPs by the moDC cells, the amount cell-associated
radioactivity was measured before and after one wash step at every
time point. Radiolabel retention was calculated as the fraction still
cell associated after washing. Cells were stained with trypan blue
and counted with Luna Cell Counting Slides (Logos biosystems, South
Korea). The measured activity per sample and the number of cells counted
were used to calculate the specific activity per cell or million cells.
5
Dual Fluorescence Cell Counting Protocol
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LUNA-FL Dual Fluorescence Cell Counter (Cat # L20001, Logos Biosystems) was used as per manufacturer’s instructions. Briefly, cells were diluted with Acridine Orange/Propidium Iodide Stain (Cat # F23001, Logos Biosystems) in a dilution of either 1:1 or 1:10 and then pipetted onto LUNA Cell Counting Slides (Cat # L12001, Logos Biosystems). Slide was loaded onto the instrument and focus was optimized using the focusing knob on the side. Every sample and its dilution were separately counted 3–4 times to get technical replicates. Total number of cells, number of live cells and number of dead cells were noted.
6
Ceramics-Induced THP-1 Cell Viability
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Adherent THP-1 cells were treated with ceramics as per the protocol above. Cells were then treated with 1 mL Accutase (Sigma-Aldrich) and incubated for 15 min in order to detach the cells from the tissue culture plate. Following incubation, the cells were collected in Eppendorf tubes and centrifuged at 5500×g for 5 min. The cell supernatant was removed, and the cells were resuspended in 200 μL complete RPMI-1640 medium. 10 μL of cell suspension was then mixed with 10 μL trypan blue (Sigma-Aldrich) and mixed gently. 10 μL of this mixed solution was then used on a Luna cell counting slide in conjunction with the Luna II (Logos Biosystems, Seoul, South Korea) fully automated cell counter in order to calculate percentage viability. Additional THP-1 cells were seeded at a density of 50,000 cells per well of a 96-well tissue culture plate and treated in accordance with the protocol above. The XTT Cell Proliferation Kit II (Sigma-Aldrich) was used according to the manufacturer’s instructions and absorbance read using a BioTek Synergy HT microplate reader at 450 nm.
7
Rab-Mediated Liposome Tethering Assay
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Fluorescence microscopy-based imaging assays for Rab-mediated liposome tethering were performed using a LUNA-FL automated fluorescence cell counter (Logos Biosystems), as described (Segawa et al., 2019 (link); Taniguchi et al., 2020 (link)). Liposomes bearing fluorescence-labeled Rh-PE or FL-PE lipids (200-nm diameter; final 2 mM total lipids) and Rab-His12 proteins (final 0.5–8 μM), which had been separately preincubated (30°C, 10 min), were mixed in RB150 with 5 mM MgCl2 and 1 mM DTT, incubated without agitation (30°C, 2 h), and then applied to a LUNA cell-counting slide (L12001, Logos Biosystems; 15 μl per well). Bright field images, Rh-fluorescence images, and FL-fluorescence images of the Rab-mediated liposome tethering reactions in the slides were obtained and processed by the LUNA-FL cell counter. Particle sizes of Rab-dependent liposome clusters observed in the fluorescence images were analyzed using the ImageJ2 software with setting the lower intensity threshold level to 150, the upper intensity threshold level to 255, and the minimum particle size to 10 pixel 2 which corresponds to approximately 10 μm2 (Segawa et al., 2019 (link); Taniguchi et al., 2020 (link)).
8
Trypan Blue Viability Assay of 3T3-L1 Adipocytes
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The effect of CK on the viability of 3T3-L1 adipocyte cells was analyzed using the Trypan blue assay. After the differentiation of 3T3-L1 pre-adipocytes and CK treatment processes were completed, as described above, the cells were collected, stained with Trypan blue, and the number of cells was measured under a microscope (Leica DM IL LED, Leica Microsystems GmbH, Wetzlar, Germany) using a Luna cell counting slide (Logos Biosystems, Anyang, Korea).
9
Fluorescence Microscopy-based Assay for Rab-mediated Liposome Tethering
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Fluorescence microscopy-based imaging assays for Rabmediated liposome tethering were performed using a LUNA-FL automated fluorescence cell counter (Logos Biosystems), as described (Segawa et al., 2019; Taniguchi et al., 2020) (link). Liposomes bearing fluorescence-labeled Rh-PE or FL-PE lipids (200-nm diameter; final 2 mM total lipids) and Rab-His12 proteins (final 0.5-8 M), which had been separately preincubated (30°C, 10 min), were mixed in RB150 with 5 mM MgCl 2 and 1 mM DTT, incubated without agitation (30°C, 2 hours), and then applied to a LUNA cell-counting slide (L12001, Logos Biosystems; 15 l per well). Bright field images, Rh-fluorescence images, and FL-fluorescence images of the Rab-mediated liposome tethering reactions in the slides were obtained and processed by the LUNA-FL cell counter. Particle sizes of Rab-dependent liposome clusters observed in the fluorescence images were analyzed using the ImageJ2 software with setting the lower intensity threshold level to 150, the upper intensity threshold level to 255, and the minimum particle size to 10 pixel 2 which corresponds to approximately 10 m 2 (Segawa et al., 2019; Taniguchi et al., 2020) (link).
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