Clone l200

Manufactured by BD

The Clone L200 is a laboratory equipment designed for general-purpose liquid handling tasks. It features a precise pipetting mechanism and a compact, user-friendly design.

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Lab products found in correlation

Amcyan, by Thermo Fisher Scientific (2 mentions) Clone fn50, by BD (2 mentions) Anti cd4 v50, by BD (2 mentions) Clone dk25, by Agilent Technologies (2 mentions) Clone l48, by BD (2 mentions) Clone 4b4 1, by Miltenyi Biotec (2 mentions) Clone l106, by BD (2 mentions) Facslyric, by BD (1 mentions) Anti cd3 percp, by BD (1 mentions) Anti cd137 pe, by Miltenyi Biotec (1 mentions) Clone rpa t8, by BD (1 mentions) Clone uchl1, by BD (1 mentions) Facscanto 2, by BD (1 mentions) Live dead fixable blue dead cell stain kit, by Thermo Fisher Scientific (1 mentions) Clone sp34 2, by BD (1 mentions) Clone b27, by BD (1 mentions)

4 protocols using clone l200

Multiparametric Flow Cytometry for SARS-CoV-2-Specific T-Cell Detection

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Detection of AIM was performed as described previously (8 (link)). Briefly, cells were stained with the following antibodies in their respective dilutions: anti–CD3-PerCP (1:25, clone SK7, BD), anti–CD4-V50 (1:50, clone L200; BD), anti–CD8–fluorescein isothiocyanate (1:25, clone DK25; Dako), anti–CD45RA-phycoerythrin (PE)–Cy7 (1:50, clone L48; BD), anti–CCR7-BV711, anti–CD69-allophycocyanin (APC)-H7 (1:50, clone FN50; BD), anti–CD137-PE (1:50, clone 4B4-1; Miltenyi), and anti–OX40-BV605 (1:25, clone L106; BD). LIVE/DEAD Fixable Aqua Dead Cell staining was included (1:100, AmCyan; Invitrogen) and acquired on a FACSLyric (BD Biosciences). After setting a time gate, LIVE CD3+ T-cells were gated, singlets were selected, and T-cells were subtyped into CD3+CD4+ and CD3+CD8+ cells. Within the CD4+ and CD8+ T-cells, T_naive were defined as CD45RA+CCR7+, T_CM as CD45RA-CCR7+, T_EM as CD45RA-CCR7-, and T_EMRA as CD45RA+CCR7-. S-specific T-cells were detected by co-expression of AIM on CD4+ (OX40 and CD137) or CD8+ (CD69 and CD137) T-cells in the combination of memory subsets (Fig. 3A). The DMSO-stimulated sample was used to set the cutoff gate for activation markers. On average, 500,000 cells were acquired per sample.

GeurtsvanKessel C.H., Geers D., Schmitz K.S., Mykytyn A.Z., Lamers M.M., Bogers S., Scherbeijn S., Gommers L., Sablerolles R.S., Nieuwkoop N.N., Rijsbergen L.C., van Dijk L.L., de Wilde J., Alblas K., Breugem T.I., Rijnders B.J., de Jager H., Weiskopf D., van der Kuy P.H., Sette A., Koopmans M.P., Grifoni A., Haagmans B.L, & de Vries R.D. (2022). Divergent SARS CoV-2 Omicron-reactive T- and B cell responses in COVID-19 vaccine recipients. Science Immunology.

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Identifying SARS-CoV-2-specific T cells

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After ex vivo stimulation of PBMCs, cells were stained at 4°C for 15 min for phenotypical lymphocyte markers and AIM expression. Surface staining was performed with the following antibodies in their respective dilutions: anti–CD3-PerCP (1:25, clone SK7, BD), anti–CD4-V50 (1:50, clone L200; BD), anti–CD8–fluorescein isothiocyanate (1:25, clone DK25; Dako), anti–CD45RA-phycoerythrin (PE)–Cy7 (1:50, clone L48; BD), anti–CCR7-BV711, anti–CD69-allophycocyanin (APC)-H7 (1:50, clone FN50; BD), anti–CD137-PE (1:50, clone 4B4-1; Miltenyi), and anti–OX40-BV605 (1:25, clone L106; BD). LIVE/DEAD Fixable Aqua Dead Cell staining was included (1:100, AmCyan; Invitrogen). Cells were first gated for LIVE CD3⁺ T cells and then subdivided into CD3⁺CD4⁺ T helper cells and CD3⁺CD8⁺ T-cytotoxic cells (Fig. 4A). SARS-CoV-2–specific T cells were identified by gating the CD69⁺CD137⁺ cells (within CD4⁺ or CD8⁺ subsets). For N = 11 of 20 donors assessed, SARS-CoV-2 specificity in the CD4⁺ subset was confirmed by gating CD137⁺OX40⁺ cells. The DMSO-stimulated sample was used to set the cutoff gate for activation markers. On average, 500,000 cells were acquired per sample. Low-frequency samples (<10,000 cells in CD4 gate and <5000 cells in CD8 gate) were excluded from analysis.

Geers D., Shamier M.C., Bogers S., den Hartog G., Gommers L., Nieuwkoop N.N., Schmitz K.S., Rijsbergen L.C., van Osch J.A., Dijkhuizen E., Smits G., Comvalius A., van Mourik D., Caniels T.G., van Gils M.J., Sanders R.W., Oude Munnink B.B., Molenkamp R., de Jager H.J., Haagmans B.L., de Swart R.L., Koopmans M.P., van Binnendijk R.S., de Vries R.D, & GeurtsvanKessel C.H. (2021). SARS-CoV-2 variants of concern partially escape humoral but not T cell responses in COVID-19 convalescent donors and vaccine recipients. Science Immunology, 6(59), eabj1750.

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T cell phenotyping and analysis

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T cells were phenotyped with antibodies against surface markers CD4 (BD PharMingen, San Jose, CA, Clone L200), CD8 (BD PharMingen, Clone RPA-T8), CD195 (BD PharMingen, Clone 3A9), CD62L (BD Biosciences, Clone SK11), and CD45RA (BD Biosciences, Clone UCHL-1) according to manufacturers’ recommendations. Live/dead discrimination was performed using propidium iodide staining. Stained cells were processed on a FACSCanto II (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR).

Paul B., Ibarra G.S., Hubbard N., Einhaus T., Astrakhan A., Rawlings D.J., Kiem H.P, & Peterson C.W. (2018). Efficient Enrichment of Gene-Modified Primary T Cells via CCR5-Targeted Integration of Mutant Dihydrofolate Reductase. Molecular Therapy. Methods & Clinical Development, 9, 347-357.

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PBMC T cell allele presentation

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PBMC T cell cultures expanded with the dominant epitope for RV1196 or Rv0125 were tested for specific allele presentation, using RM3 cells transfected with MHC II alleles at a 1:1 or 2:1 ratio (i.e., T cell:transfected RM3 cell) (Fig. S1). Transfected RM3 cells were prepared for T cell testing via the addition of 1 μg/mL peptide to individual wells of a 96-well plate and were incubated for 90 min at 37°C and 5% CO₂. Following the incubation, the prepared T cells were added to each well of a 96-well plate and incubated for another 12 h in the presence of a Golgi plug inhibitor, namely, BFA (BD Biosciences, catalog number 555029). Stimulation plates were spun down and washed with 1× PBS, following 12 h of incubation. Flow cytometry staining was performed for viability (Live/Dead Fixable Blue Dead Cell Stain Kit, Thermo Fisher, catalog number L34962) and surface marker expression for CD3 (BD, clone SP34-2), CD4 (BD, clone L200), CD8 (BD, clone RPA-T8), HLA DR/DP/DQ (Bio-Rad clone Bu26; Beckman clone I3; BioLegend, clone Tü39), and the intracellular cytokines IFN-γ (BD, clone B27) and TNF (BD, clone Mab11), using standard flow cytometry and intracellular staining protocols. The samples were run on a BD LSRII or a Cytek Aurora (Cytek, Bethesda, MD, USA) and were analyzed using the FlowJo software package (BD, version 10).

Grant N.L., Kelly K., Maiello P., Abbott H., O’Connor S., Lin P.L., Scanga C.A, & Flynn J.L. (2023). Mycobacterium tuberculosis-Specific CD4 T Cells Expressing Transcription Factors T-Bet or RORγT Associate with Bacterial Control in Granulomas. mBio, 14(3), e00477-23.

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