Human NK cell lines, NK-92 (ATCC #CRL-2407) and PTA-6967 (ATCC #PTA-6967) were maintained in NK cell media as described previously (12 (link)). NK cell media [Alpha Minimum Essential medium without ribonucleosides and deoxyribonucleosides but with 2 mM L-glutamine and 1.5 g/L sodium bicarbonate (Gibco #12561-056) was supplemented with 0.2 mM inositol, 0.1 mM 2-mercaptoethanol, 0.02 mM folic acid, 12.5% horse serum (Gibco #16050-122), 12.5% fetal calf serum (Gibco #10438-034), and 100 U/mL recombinant IL-2 (R&D Systems #202-IL)].
Pta 6967
Manufactured by American Type Culture Collection
The PTA-6967 is a laboratory equipment piece designed for cell culture applications. It is a benchtop centrifuge capable of separating cellular components and other biological materials. The device operates using a rotor system to spin samples at controlled speeds and durations to facilitate various cell culture and sample preparation workflows.
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Lab products found in correlation
Vero c1008, by American Type Culture Collection (2 mentions)
Permeabilization buffer, by BioLegend (2 mentions)
Clone b27, by BD (2 mentions)
Anti cd107a clone h4a3, by BD (2 mentions)
Anti cd56 clone 5.1h11, by BD (2 mentions)
Anti cd16 clone 3g8, by BD (2 mentions)
Brefeldin a, by BioLegend (2 mentions)
Golgistop, by BD (2 mentions)
Lsrfortessa, by BD (2 mentions)
Clone mab11, by BD (1 mentions)
Nr 52309, by BEI Resources (1 mentions)
Atcc tib 202, by American Type Culture Collection (1 mentions)
5 protocols using pta 6967
1
Expansion and Maintenance of NK Cell Lines
2
Cell Culture Protocols for Viral Studies
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Vero E6 cells were purchased from ATCC (ATCC VERO C1008), grown at 37C, 5% CO2 and maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% non-essential amino acids. THP-1 cells were purchased from ATCC (ATCC TIB-202), grown at 37C, 5% CO2 and maintained in RPMI-1640 supplemented with 10% fetal bovine serum, 2mM L-glutamine, 10mM HEPES, and 0.05 mM β-mercaptoethanol. CD16.NK-92 (ATCC PTA-6967) were purchased from ATCC (ATCC PTA-6967), grown at 37C, 5% CO2 and maintained in in MEM-α supplemented with 12.5% FBS, 12.5% horse serum, 1.5g/L sodium bicarbonate, 0.02mM folic acid, 0.2mM inositol, 0.1 mM 2-β-mercaptoethanol, 100U/mL recombinant IL-2.
3
ADNKA Assay for SARS-CoV-2 RBD Antibodies
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ADNKA assay was performed as described with modifications (Gunn et al., 2020 (link)). ELISA plates were coated with recombinant RBD antigen (300 ng/well) (Bates et al., 2021c (link)) (BEI Resources NR-52309). Wells were washed, blocked, and incubated with serial dilutions of sera (1:10, 1:30, 1:90) for 2hrs at 37°C prior to adding CD16a.NK-92 cells (PTA-6967, ATCC) (5 × 104 cells/well) for 5hrs with brefeldin A (Biolegend), Golgi Stop (BD Biosciences) and anti-CD107a (clone H4A3, BD Biosciences). Cells were stained with anti-CD56 (clone 5.1H11, BD Biosciences) and anti-CD16 (clone 3G8, BD Biosciences) and fixed with 4% PFA. Intracellular cytokine staining to detect IFNγ (clone B27, BD Biosciences) and TNFα (clone Mab11, BD Biosciences) was performed in permeabilization buffer (Biolegend). Markers were measured using a BD LSRFortessa and analyzed by FlowJo10. CD16 expression was confirmed in all cells. NK cell degranulation and activation were calculated as percent of CD56+NK cells positive for CD107a, or IFNγ or TNFα expression. Representative data from one dilution was chosen by the highest signal to noise ratio for further analyses.
4
Evaluating NK Cell Activation by SARS-CoV-2 RBD
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ADNKA was performed as described (26 (link), 133 (link)). ELISA plates were coated with recombinant RBD (300 ng/well) (BEI Resources NR-52309). Wells were washed, blocked, and incubated with serially diluted samples (1:10, 1:100, 1:1000) in duplicate for 2hours at 37°C prior to adding CD16a.NK-92 cells (PTA-6967, ATCC) (5 × 104 cells/well) for 5hours with brefeldin A (Biolegend), Golgi Stop (BDBiosciences) and anti-CD107a (clone H4A3, BDBiosciences). Cells were stained with anti-CD56 (clone 5.1H11, BDBiosciences) and anti-CD16 (clone 3G8, BDBiosciences) and fixed with 4%PFA. Intracellular cytokine staining to detect IFNγ (clone B27, BDBiosciences) and TNFα (clone Mab11, BDBiosciences) was performed in permeabilization buffer (Biolegend). Markers were measured using a BD LSRFortessa and analyzed by FlowJo10. CD16 expression was confirmed. NK cell degranulation and activation were calculated as %CD56+CD107a+, IFNγ+ or TNFα+. Representative data from one dilution was chosen by the highest signal-to-noise ratio. Experiments were conducted two independent times.
5
Cell Culture of Vero E6, THP-1, and CD16.NK-92
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Vero E6 cells were purchased from ATCC (ATCC VERO C1008), grown at 37C, 5% CO2 and maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% non-essential amino acids. THP-1 cells were purchased from ATCC (ATCC TIB-202), grown at 37C, 5% CO2 and maintained in RPMI-1640 supplemented with 10% fetal bovine serum, 2mM L-glutamine, 10mM HEPES, and 0.05 mM β-mercaptoethanol. CD16.NK-92 were purchased from ATCC (ATCC PTA-6967), grown at 37C, 5% CO2 and maintained in in MEM-α supplemented with 12.5% FBS, 12.5% horse serum, 1.5g/L sodium bicarbonate, 0.02mM folic acid, 0.2mM inositol, 0.1 mM 2-β-mercaptoethanol, 100U/mL recombinant IL-2.
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