HeLa cells were grown in Gibco™ DMEM Medium (1x Dulbecco’s Modified Eagle Medium; ThermoFisher Scientific, Cat. No. 31885-023) including 10% Fetal Bovine Serum (Biochrom AG; Cat. No. S0415), 1% MEM NEAA (100x) (Minimum Essential Medium Non-Essential Amino Acid; ThermoFisher Scientific; Cat. No. 11140-035) and 1% Penicillin-Streptomycin (Sigma; Cat. No. P0781) overnight at 37 °C. Jurkat cells are grown in Gibco™ RPMI Medium 1640 (1x) (Thermo Fisher Scientific; Cat. No. 31870-025) including 10% Fetal Bovine Serum (Biochrom AG; Cat. No. S0415), 1% MEM NEAA (100x) (Minimum Essential Medium Non-Essential Amino Acid; ThermoFisher Scientific; Cat. No. 11140-035) and 1% Penicillin-Streptomycin (Sigma; Cat. No. P0781). After aspiration of medium, and PBS wash, HeLa cells are incubated in Trypsin/EDTA (Sigma; Cat. No. T3924) for 5–10 min at 37 °C followed by addition of medium to stop trypsination of cells. After centrifugation (Hettich Rotina 380 R; 5 min × 300 g) of HeLa or Jurkat cells and an additional washing in PBS, cells are counted in Counting Slides (BIO RAD; Cat. No. 145-0011) in TC20 Automated Cell Counter (BIO RAD) by staining with trypan-blue (BIO RAD; Cat. No. 145-0013). In general, a fraction of >80% of viable cells were determined. After counting, cells were diluted to the intended cell number per sample.
Counting slide
Manufactured by Bio-Rad
Sourced in United Kingdom
The Counting Slides are a versatile laboratory equipment used for the enumeration and analysis of cells, particles, or other microscopic entities. The slides provide a standardized platform for consistent and precise counting, enabling accurate quantification of samples.
Automatically generated - may contain errors
Lab products found in correlation
Tc20 automated cell counter, by Bio-Rad (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Trypan blue, by Bio-Rad (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Mem neaa, by Thermo Fisher Scientific (1 mentions)
Mem neaa 100, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Trypan blue solution, by Bio-Rad (1 mentions)
Trypan blue solution, by Merck Group (1 mentions)
T75 flask, by BD (1 mentions)
1640 medium, by Thermo Fisher Scientific (1 mentions)
U937 cells, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Trypan blue dye, by Bio-Rad (1 mentions)
Penicillin streptomycin p s, by Merck Group (1 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
9 protocols using counting slide
1
Detailed Cell Culture Protocols
2
Culturing Human Astrocytoma Cells (1321N1)
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The human astrocytoma cell line (1321N1), established by Macintyre [40 (link)], was purchased from the European Collection of Cell Cultures (Wiltshire, UK). The 1321N1 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin–streptomycin (Thermo Fisher Scientific). Cells were maintained at 37 °C in a humidified incubator under 5% CO2 atmosphere. The 1321N1 cells were divided into the vehicle group and other groups with or without several treatments mentioned later. The number of cells was counted by the trypan blue dye exclusion method using a TC20™ automated cell counter (Bio-Rad Laboratories, Hercules, CA, USA) on counting slides (Bio-Rad).
3
Trypan Blue Viability Assay
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Cell survival was assessed by trypan blue staining and counting. Cells were detached with accutase 20 to 24 hours after stimulation and mixed with the equal volume of 0.4% trypan blue solution (Bio‐Rad, Munich, Germany). The suspension was transferred to counting slides (Bio‐Rad), and total as well as living cell number was analyzed automatically by using the TC20 Automated Cell Counter (Bio‐Rad). Cell viability was calculated by the ratio of living to total cell count, and all values were normalized to cell viability of control cells.
4
Cell Proliferation Assay Protocol
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1 x 105 cells were plated in triplicate in 6-well plates for proliferation. After 48 hours, cells were lifted up and mixed with Trypan Blue solution (Sigma, #T8154) at 1:1 ratio. Then, cells were placed on counting slides (Bio-Rad, #145–0011) and counted using a Bio-Rad TC20 Automated Cell Counter (Bio-Rad, #1450102).
5
Culturing U937 Monocyte Cell Line
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U937 cells (human monocyte cell line) were obtained from ATCC (LGC Standards, UK) and maintained in complete Roswell Park Memorial Institute (RPMI) – 1640 Medium (Gibco, UK) containing 10% fetal bovine serum, 5 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, and 10 mM (4-(2-hydroxyethyl)-1-piperazineethanosulfonic acid (HEPES) buffer (all Sigma-Aldrich, UK) in a humidified CO2 incubator (5% CO2 and 37 °C). U937 cells were grown and propagated in T75 flasks (Falcon, UK). Cells were maintained at 2-3 million cells/mL density. Only cultures with at least 90% viability were used in experiments. Cell viability and cell counts were obtained using trypan blue exclusion method wherein equal volumes of cells and 0.4% trypan blue (Bio-Rad, UK) were mixed, placed on counting slides (Bio-Rad, UK), then counted using an automated cell counter (TC20, Bio-Rad, UK).
6
Apoptosis Assay in Breast, Neuroblastoma, and Stem Cells
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We used the human breast tumor cell line MCF-7 obtained from ATCC s , the neuroblastoma cell line N2a (overexpressing a-synuclein, N2a a-Syn, expressing together with mCherry as a marker 17 ) and the H13 human embryonic stem cell line.
DMEM Media -GlutaMAXt, DMEM/F12, Fetal Bovine Serum (FBS), KSR, L-glutamine, Dispase, Accutase, Phosphate-Buffered Saline pH 7.4 (1Â PBS), SYTOX s Red Dead Cell Stain and Annexin V Alexa Flour s 488 conjugates were purchased from Life Technologies. Counting slides and the trypan blue dye were obtained from BIO-RAD, UK. 10Â Phosphate-Buffered Saline (10Â PBS), Trypsin, Penicillin-Streptomycin (PS), Staurosporine (STS), and Dimethyl Sulfoxide (DMSO) were purchased from Sigma Aldrich. NEAA and donkey serum were purchased from Millipore and FGF2 was purchased from Peprotech.
DMEM Media -GlutaMAXt, DMEM/F12, Fetal Bovine Serum (FBS), KSR, L-glutamine, Dispase, Accutase, Phosphate-Buffered Saline pH 7.4 (1Â PBS), SYTOX s Red Dead Cell Stain and Annexin V Alexa Flour s 488 conjugates were purchased from Life Technologies. Counting slides and the trypan blue dye were obtained from BIO-RAD, UK. 10Â Phosphate-Buffered Saline (10Â PBS), Trypsin, Penicillin-Streptomycin (PS), Staurosporine (STS), and Dimethyl Sulfoxide (DMSO) were purchased from Sigma Aldrich. NEAA and donkey serum were purchased from Millipore and FGF2 was purchased from Peprotech.
7
Evaluating iPSC Editing and Selection
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iPSC lines edited at the HPRT1 locus and controls were plated at 3 × 104 cells per well in a 6-well culture dish, grown for 2 days without HAT, followed by 2 additional days with or without HAT. Cells were harvested on days 2, 3, and 4 post-plating, and re-suspended in 100 µL of AK02N. An 11 μL aliquot of cell suspension was mixed 1:1 with Trypan Blue Stain 0.4% (Gibco) by gentle pipetting, and 10 μL was applied to each side of a Counting Slide (Bio-Rad). Cell numbers were determined with the TC20 Automated Cell Counter (Bio-Rad).
iPSC lines edited at the APRT locus and controls were plated at 1 × 105 cells per well in a 6-well culture dish in media containing Y-27632 and grown for 2 days. 2, 6-diaminopurine (DAP; Sigma) dissolved in DMSO was added to growth media at a final concentration of 10 µg/mL for an additional 2 days, after which the wells were stained with crystal violet and imaged. Treatment with DMSO was used as a control.
iPSC lines edited at the APRT locus and controls were plated at 1 × 105 cells per well in a 6-well culture dish in media containing Y-27632 and grown for 2 days. 2, 6-diaminopurine (DAP; Sigma) dissolved in DMSO was added to growth media at a final concentration of 10 µg/mL for an additional 2 days, after which the wells were stained with crystal violet and imaged. Treatment with DMSO was used as a control.
8
Trypan Blue Cell Viability Assay
9
MIA PaCa-2 Cell Proliferation Assay
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A total of 5 × 104 MIA PaCa-2 cells were seeded in triplicate in 12-well plates. After 48 h, cells were trypsinized for the counting. Cells were placed on the counting slide (Bio-Rad, #145-0011) and counted using a Bio-Rad TC20 Automated Cell Counter (Bio-Rad, #1450102). For SNAT7 WT, SNAT7 KO, HA-SNAT7WT, HA-SNAT7N62H, and HA-SNAT721-C cell proliferation, 5 × 104 cells were plated in triplicate in 12-well plates. After 24 h, cells were stimulated in 0.2 mM glutamine (Sigma, #G3126) medium (Thermo Fisher Scientific, #11960069) with or without 3% BSA (Sigma, #A1470) and 500 nM Torin (Sigma, #475991) and were incubated for 48 h. Then, cells were washed three times with 1× PBS and stained with 0.5% crystal violet (Sigma, #C6158) in 20% methanol. For measuring absorbance at 590 nm, stained cells were resolved in ethanol. Cells incubated for 24 h in low glutamine were used as a basal level.
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