OT-I CD8+ T cells were co-cultured with either tumor cells or tumor expressing H-2Kb-OVA257-264 cells for 2 and 48 h. In the 2 h test, cells were co-cultured at the presence of anti-CD107a (Biolegend, 1D4B). After 2h, all cells were collected and stained with anti-CD8a (Biolegend) for degranulation analysis via flow cytometry (Cytoflex, Beckman). After 48 h, all cells were collected and stained with anti-CD8a, PI and Annexin V (Biolegend) for target cell apoptosis analysis via flow cytometry (Cytoflex, Beckman). All FCM data were analyzed by Flowjo 10.
Anti cd107a
Manufactured by BioLegend
Sourced in Denmark, United States
Anti-CD107a is a laboratory reagent used for the detection and analysis of CD107a expression on the cell surface. CD107a, also known as LAMP-1, is a lysosome-associated membrane glycoprotein that is often used as a marker for degranulation in immune cells such as natural killer cells and cytotoxic T cells. This reagent can be used in flow cytometry and other cell-based assays to measure the activation and function of these cell types.
Automatically generated - may contain errors
Lab products found in correlation
Golgiplug, by BD (6 mentions)
Golgistop, by BD (4 mentions)
Anti cd69, by BioLegend (4 mentions)
Anti ifn γ, by BioLegend (4 mentions)
Anti cd8, by BioLegend (4 mentions)
Phorbol myristate acetate (pma), by Merck Group (3 mentions)
Ionomycin, by Merck Group (3 mentions)
Anti cd3, by BioLegend (3 mentions)
Anti tigit, by BioLegend (2 mentions)
Anti tim3, by Thermo Fisher Scientific (2 mentions)
Anti cd107b, by BioLegend (2 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Avantor (2 mentions)
Cytofix fixation buffer, by BD (2 mentions)
Aurora spectral cytometer, by Cytek (2 mentions)
Anti nkg2d, by BioLegend (2 mentions)
Anti pd 1, by BioLegend (2 mentions)
Anti tim 3, by BioLegend (2 mentions)
Anti tnf α, by BioLegend (2 mentions)
Lsrfortessa, by BD (2 mentions)
26 protocols using anti cd107a
1
OT-I CD8+ T Cell Cytotoxicity Assay
2
Comprehensive Immune Profiling of CAR-T Cells
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The following mAbs were used in this study; anti‐CD8α, anti‐TIGIT, anti‐CD107a, and anti‐Bcl‐2 (BioLegend), anti‐CD90.2, anti‐PD‐1, anti‐LAG‐3, anti‐TIM‐3, and anti‐Vα8.3 mAb (Thermo Fisher Scientific), antihuman CD20 (BD Biosciences), anti‐Bcl‐xL (Abcam), and anti‐Bim (CST). Annexin V (BD Biosciences) and Zombie Yellow viability dye (BioLegend) were also used. Biotinylated recombinant protein L (GenScript) was used to detect CAR‐T cells, as previously reported.16 Intracellular protein staining buffer set (Thermo Fisher Scientific) was also used in some experiments. APC‐conjugated antihuman IgG mAb (BioLegend) was used to detect the binding of mouse PD‐L1‐human Fc fusion protein (R&D Systems). Antimouse CD16/CD32 mAb was used for blockade of non‐specific binding of mAb to Fcγ receptors. Flow cytometric data were acquired by EC800 (SONY) or BD LSRFortessa X‐20 cell analyzer (BD Biosciences), and analyzed using FlowJo software (FlowJo, LLC).
3
Characterization of Tumor-Infiltrating Lymphocytes
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Purified CD45+ TIL were co-cultured for 4 h with B16F10 tumour cells, pulsed with Trp2 (1 µg) and gp100 (1 µg) peptides, in the presence of Brefeldin A (Invitrogen 00-4506-51, 1/1000), monensin (Merck M5273, 2 µm ) and anti-CD107a mAb (clone 1D4B, Biolegend 121619, 1/50). TIL were stained with mAb specific for surface proteins prior to fixation and permeabilization. Permeabilized cells were then stained with anti-IFNγ (clone XMG1.2, Biolegend 505808, 1/200), anti-TNF (clone MP6-XT22, Biolegend 506321, 1/100) and anti-perforin (clone eBioOMAK-D, eBioscience 17-9392-80, 1/50) mAb.
For cytotoxicity experiments, freshly isolated CD8+ TIL were either kept in medium or pretreated with neutralising anti-Nrp-1 (clone 761704, R&D systems MAB59941, 10 µg/ml), anti-PD-1 (clone RMP1-14, Bio-X-Cell BE0146, 10 µg/ml) or isotype controls (clone 2A3, Bio-X-Cell BE0089, 10 µg/ml). Cytotoxic activity toward the B16F10 cell line, pulsed with Trp2 and gp100 peptides, or the MC-38 cell line was evaluated using a conventional 4 h and overnight chromium (51Cr) release assay.
For experiments with human freshly isolated lung TIL and CTL clone P62, activated T cells were incubated for 30 min to 1 h either with bovine serum albumin (BSA, Merck) or Sema-3A-Fc (R&D systems 1250-S3-025, 100 ng/ml), and their cytotoxic activity toward the autologous tumour cell was evaluated.
For cytotoxicity experiments, freshly isolated CD8+ TIL were either kept in medium or pretreated with neutralising anti-Nrp-1 (clone 761704, R&D systems MAB59941, 10 µg/ml), anti-PD-1 (clone RMP1-14, Bio-X-Cell BE0146, 10 µg/ml) or isotype controls (clone 2A3, Bio-X-Cell BE0089, 10 µg/ml). Cytotoxic activity toward the B16F10 cell line, pulsed with Trp2 and gp100 peptides, or the MC-38 cell line was evaluated using a conventional 4 h and overnight chromium (51Cr) release assay.
For experiments with human freshly isolated lung TIL and CTL clone P62, activated T cells were incubated for 30 min to 1 h either with bovine serum albumin (BSA, Merck) or Sema-3A-Fc (R&D systems 1250-S3-025, 100 ng/ml), and their cytotoxic activity toward the autologous tumour cell was evaluated.
4
LCMV-specific CD8+ T cell Characterization
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Mice were infected with 2 x 105 PFU of the LCMV Armstrong strain intraperitoneally (i.p.) (27 (link)). Eight days after infection mice were euthanized, and the single cell suspensions of splenocytes were prepared as previously described (11 (link)). Virus-specific CD8+ T cells were identified with R-phycoerythrin (PE) or allophycocyanin (APC)-labeled H2-Db/GP33-41 and H2-Db/NP396-404 dextramers (Immudex) and brilliant violet (BV) 421-labled H2-Db/GP33-41 tetramers (kindly provided by the NIH Tetramer Core Facility). For the detection of cytokine production, splenocytes were incubated with 1 μM GP33 (KAVYNFATC) peptides (Anaspec) in the presence of GolgiStop (BD Biosciences) and GolgiPlug (BD Biosciences) for 4.5 hours at 37°C. In order to detect CD107a and CD107b surface externalization, anti-CD107a (1:1000; Biolegend) and anti-CD107b (1:1000; Biolegend) antibodies were added during peptide stimulation.
5
Intracellular Granzyme B Assay in PBMCs
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PBMCs were stimulated initially for 5 h in complete media with 50 ng/ml phorbol‐12‐myristate‐13‐acetate (PMA; Sigma) and 1 μg/ml ionomycin (Sigma) in the presence of anti‐CD107a (BioLegend) at 37°C in 5% CO2. After 1 h of stimulation, brefeldin (10 μg/ml) and monensin (2 μM) were added and were present for the last 4 h of culture. Cells were vortexed periodically to prevent cell settling. PBMCs were then washed and stained for cell‐surface markers as described previously. Finally, cells were fixed with intracellular (IC) fixation buffer (eBioscience) and permeabilized and stained for detection of intracellular granzyme B, as described above.
6
Multiparameter Flow Cytometry Analysis of Immune Cell Populations
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The following antibodies were used (all purchased from BioLegend): anti-CD45 (30-F11, Alexa Fluor 700), anti-CD3ε (145-2C11, APC-Cy7, PE-Cy5), anti-CD4 (GK1.5, BV421), anti-CD8a (53-6.7, BV650), anti-CD19 (6D5, PE-Cy5), anti-Ter119 (TER-119, PE-Cy5), anti-F4/80 (BM8, PE-Cy5), anti-NKp46 (29A1.4, PerCP-Cy5.5), anti-NK1.1 (PK136, BV711, FITC), anti-CD69 (H1.2F3, BV605), anti-Sca1 (D7, BV510), anti-Ki67 (SolA15, eFluor 450), anti-CD107a (1D4B, Alexa Fluor 647), and anti-IFNγ (XMG1.2, PE). Anti–granzyme B (GB11, PE-CF594) was purchased from BD Biosciences.
7
Multiparametric Flow Cytometry Analysis
8
Intracellular Cytokine Staining Assay
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Frozen cells were thawed as described in “Processing for CITE-seq.” 2 million PBMC, adenoid, or tonsil cells from each sample were resuspended in 200 μL of complete RPMI medium containing 10% FBS (VWR), 2 mM glutamine, 0.055 mM beta-mercaptoethanol, 1% penicillin/streptomycin, 1 mM sodium pyruvate, 10 mM HEPES, and 1% non-essential amino acids. Cells were stimulated with PMA (50ng/ml, Sigma) and ionomycin (1000ng/ml, Sigma) for 2.5 h in the presence of anti-CD107a (BioLegend), GolgiSTOP (monensin, BD), and GolgiPlug (BFA, BD). After stimulation, surface markers were stained as described above in the AIM assay. Surface-stained cells were washed and fixed with Cytofix Fixation Buffer (BD) at RT for 20 min and washed with permeabilization buffer (eBioscience) twice. Then, the intracellular cytokine antibody mix was added for 30 min at RT (staining volume 50uL). Stained cells were collected on the Aurora spectral cytometer (Cytek). Antibodies used in this assay are listed in Supplementary Table 8 .
9
Cytokine Release Assay for NK Cells
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NK effector cells were plated alone or with target K562 cells at a 2:1 ratio. The cells were incubated with anti-CD107a (BioLegend 328606, RRID:AB_1186036) at 37°C and 5% CO2. After 1 h, cytokine release was inhibited with Golgi Stop (BD Biosciences 554724, RRID:AB_2869012) and Golgi Plug (BD Biosciences 555029, RRID:AB_2869014). The cells were incubated together for an additional 4 h. At collection, surface staining for CD3 (BioLegend 317330, RRID:AB_2563507), CD56 (BioLegend 92189), and CD45 (BioLegend 304042, RRID:AB_2562106) was performed followed by fixation with 2% paraformaldehyde and permeabilization with 0.1% Triton X. Intracellular staining was then completed for INFγ (BioLegend 93705) and TNFα (BioLegend 92960). Samples were analysed with a LSR II instrument (BD Biosciences) with 20 mW Blue (488 nm), 40 mW Red (640 nm), 25 mW Violet (405 nm) lasers and data analysed by FlowJo version v10.8.1_CL (BD Life Sciences) [81 ].
10
Comprehensive NK Cell Phenotyping
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The following fluorescently conjugated antibodies were used for phenotypic analysis of NK cells: anti-LFA-1 (363404); anti-CXCR3 (353720); anti-PD-1 (329906); anti-CD107a (328612); anti-NKP44 (325116); anti-CD158e1 (312706); anti-CD158d (347006); anti-CD27 (356412); anti-CCR4 (359412); anti-DNAM-1 (338316); anti-CD16 (302040); anti-Granzyme B (515406); anti-CD62L (304806); anti-CD69 (310910); anti-NKP80 (346706); anti-NKP30 (325210); anti-CD158f (341304); anti-CD158b (312612); anti-Tim-3 (345012); anti-CD94 (305504); anti-TIGIT (372706); anti-TRAIL (308206); anti-CD57 (322306); anti-CX3CR1 (341610); anti-NKG2D (320808); anti-Perforin (353310); anti-Ki67 (350504); anti-IFN-γ (506518); anti-CD94 (305506); anti-NKp46 (331916); anti-CTLA4 (369614); anti-CD96 (338416); anti-41BB (309818); anti-CD25 (356108) were purchased from Biolegend. anti-CD159a/NKG2A (FAB1059P) and anti-NKG2C (FAB138G) were purchased from R&D.
The following fluorescently conjugated antibodies were used to identify immune cell types in eNK or PBMC: anti-human Lineage Cocktail (348803); anti-CD56 (362550); anti-CD3 (300430); anti-CD33 (366620); anti-HLA-DR (307606); anti-CD14 (301836); anti-CD19 (302242); anti-CD11b (301322); anti-CD25 (356108); and anti-FOXP3 (320108) were purchased from Biolegend, San Diego, CA, USA.
The following fluorescently conjugated antibodies were used to identify immune cell types in eNK or PBMC: anti-human Lineage Cocktail (348803); anti-CD56 (362550); anti-CD3 (300430); anti-CD33 (366620); anti-HLA-DR (307606); anti-CD14 (301836); anti-CD19 (302242); anti-CD11b (301322); anti-CD25 (356108); and anti-FOXP3 (320108) were purchased from Biolegend, San Diego, CA, USA.
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