Blood was collected from each animal in heparinised vacutainer tubes (Ref: 368480, BD). PBMCs were isolated using the Ficoll PaqueTM (Ref: 17.1440.03, GE Healthcare, Uppsala, Sweden) density gradient centrifugation technique. Briefly, the heparinised blood was mixed with an equal volume of sterile PBS, layered on Ficoll Paque and centrifuged at 1650× g for 30 min at RT with no brakes. The interphase layer containing PBMCs was collected, topped up with sterile 1xPBS and spun at 670× g for 10 min with brakes on. The pellet was resuspended in 10 mL of tris-ammonium chloride and incubated in a water bath set at 37 °C for 10 min to lyse residual RBC. The cell suspension was spun at 300× g for 10 min to remove residual platelets. The cell pellet was washed twice by suspending in 1xPBS and spun at 300× g for 10 min. After washing, PBMCs were re-suspended in complete RPMI1640 ((Ref: R4130-10L, Sigma) supplemented with 10% foetal bovine serum (Ref: A4766, GIBCO), 1% L-glutamine (Ref: 21051040, Thermo Fisher Scientific) and 1% penicillin/streptomycin (Ref: HP10.1, Roth).
Penicillin streptomycin
Manufactured by Carl Roth
Sourced in Germany
Penicillin/streptomycin is a commonly used antibiotic solution for cell culture applications. It contains the antibiotics penicillin and streptomycin, which act to inhibit the growth of bacteria in cell culture media.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (3 mentions)
Ficoll paque, by GE Healthcare (2 mentions)
Dimethyl sulfoxide (dmso), by Carl Roth (2 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Vacutainer tube, by BD (1 mentions)
Pbs solution, by Merck Group (1 mentions)
Trypsin edta, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Ccl 86, by American Type Culture Collection (1 mentions)
Hut78, by American Type Culture Collection (1 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (1 mentions)
Rpmi 1640 medium, by Lonza (1 mentions)
L glucose, by Biosynth (1 mentions)
L glutamine, by Biosynth (1 mentions)
Mercaptoethanol, by Carl Roth (1 mentions)
Lysogeny broth medium, by Carl Roth (1 mentions)
Isopropyl β d thiogalactopyranoside iptg, by Carl Roth (1 mentions)
Hepes, by Thermo Fisher Scientific (1 mentions)
6 protocols using penicillin streptomycin
1
Isolation of Peripheral Blood Mononuclear Cells
2
Culturing Human Breast Cell Lines
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The epithelial mammary human cell line HBL-100 used in this work as normal cells and invasive ductal carcinoma BT-20 were received by our laboratory from CLS Cell Lines Service GmbH (Eppelheim, Germany). Cells were cultured at the first stage in a Petri flask in Dulbecco's modified Eagle's medium (DMEM) obtained from Merck KGaA (Darmstadt, Germany) containing 10% FBS (Merck KGaA, Darmstadt, Germany) and 1% penicillin/streptomycin (Carl Roth GmbH + Co. KG, Karlsruhe Germany). After approximately 72 hours Trypsin/EDTA (Merck KGaA, Darmstadt, Germany) was used to detach the cells, and they were subsequently centrifuged and transferred onto plasma treated glass coverslips inside plastic Petri dishes and incubated at 37 °C/5% CO2. For reaching a large confluence of cells on the coverslips, cells were fed 1–2 times before the AFM experiment, during which both samples were washed by PBS solution (Merck KGaA, Darmstadt, Germany) each time to change the culture medium.
3
PBMC Isolation from Whole Blood
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Peripheral blood mononuclear cells (PBMCs) were isolated from 20 mL of blood collected in heparinized vacutainer tubes (Ref: 368480, BD) using the Ficoll-PaqueTM (GE Healthcare, Uppsala, Sweden) density gradient centrifugation. Briefly, heparinized blood was mixed with an equal volume of sterile PBS, layered on Ficoll-Paque, and centrifuged at 1650× g for 30 min at room temperature with no brakes. The interphase layer containing PBMCs was harvested, washed with sterile 1 × PBS, and spun at 670× g for 10 min with brakes on. The pellet was resuspended in 10 mL of tris-ammonium chloride and incubated in a water bath set at 37 °C for 10 min to lyse residual RBCs. The cell suspension was spun at 300× g for 10 min to remove residual platelets. The cell pellet was washed twice by suspending in 1 × PBS and spun at 300× g for 10 min. Isolated PBMCs were 40 µm-filtered to remove fat and resuspended in 20 mL of RPMI medium supplemented with 10% fetal bovine serum (Ref: A4766, GIBCO), 1% L-glutamine (Ref: 21051040, Thermo Fisher Scientific, Waltham, MA, USA), and 1% Penicillin/Streptomycin (Ref: HP10.1, Roth). PBMCs were counted in a Neubauer chamber using the Trypan Blue dye exclusion method.
4
Cell Culture Conditions for HEK293T, Raji, J-Lat, and HuT78
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HEK293T/17 (CRL-11268, ATCC) cells were maintained in DMEM (Lonza) supplemented with 10% (v/v) heat-inactivated FCS (fetal calf serum, Biochrom) and 2 mM l -glutamine. Raji (CCL-86, ATCC) and J-Lat_8.4 (NIH 9847, obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: J-Lat Full Length Clone 8.4 from Dr. Eric Verdin) cells were cultivated in RPMI1640 medium (Lonza) containing 10% (v/v) FCS and 2 mM l -glutamine. The same medium was applied for human primary T cells, but with additional 200 U/ml penicillin/streptomycin (Carl Roth; T cell medium). RPMI1640 used for cell line HuT78 (TIB-161, ATCC) contained 20% (v/v) FCS and 2 mM l -glutamine. All cell lines were tested and negative for mycoplasma.
5
Antibody Staining and Cell Culture Protocol
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The following antibodies were purchased from BioLegend (San Diego, CA, USA): Alexa FluorTM 647 αHisTag Antibody (Cat. No.: 652513) and Purified anti-HisTag Antibody (Cat. No.: 652501). Peroxidase AffiniPure Donkey αMouse IgG (H + L) antibody (Cat. No.: 715-035-151) was bought from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA).
Roswell Park Memorial Institute 1640 medium (RPMI 1640), fetal bovine serum (FBS), L-Glutamine, HEPES, and 0.05% Trypsin-EDTA (1×) were commercially bought from Thermo Fisher Scientific (Waltham, MA, USA). DMSO, Isopropyl-β-D-thiogalactopyranoside (IPTG), penicillin/streptomycin, ß-mercaptoethanol, ampicillin, and lysogeny broth (LB)-medium were purchased from Carl Roth GmbH (Karlsruhe, Germany). PBS, Pancoll, and Minimum Essential Medium (MEM) Non-Essential Amino Acid Solution (100×) were obtained from PAN Biotech (Aidenbach, Germany). Dulbecco´s Modified Eagle´s Medium (DMEM) was provided by Capricorn Scientific GmbH (Ebsdorfergrund, Germany). The DMEM was already supplied with 1.0 g/L L-glucose, 1.0 g/L L-Glutamine, 1.0 g/L sodium pyruvate, and 3.7 g/L NaHCO3 D-Luciferin Firefly was obtained from Biosynth (Staad, Switzerland).
Roswell Park Memorial Institute 1640 medium (RPMI 1640), fetal bovine serum (FBS), L-Glutamine, HEPES, and 0.05% Trypsin-EDTA (1×) were commercially bought from Thermo Fisher Scientific (Waltham, MA, USA). DMSO, Isopropyl-β-D-thiogalactopyranoside (IPTG), penicillin/streptomycin, ß-mercaptoethanol, ampicillin, and lysogeny broth (LB)-medium were purchased from Carl Roth GmbH (Karlsruhe, Germany). PBS, Pancoll, and Minimum Essential Medium (MEM) Non-Essential Amino Acid Solution (100×) were obtained from PAN Biotech (Aidenbach, Germany). Dulbecco´s Modified Eagle´s Medium (DMEM) was provided by Capricorn Scientific GmbH (Ebsdorfergrund, Germany). The DMEM was already supplied with 1.0 g/L L-glucose, 1.0 g/L L-Glutamine, 1.0 g/L sodium pyruvate, and 3.7 g/L NaHCO3 D-Luciferin Firefly was obtained from Biosynth (Staad, Switzerland).
6
Comprehensive Immunolabeling Protocol
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The following antibodies were
used: Alexa Fluor 647-labeled anti-6-His epitope tag (Cat. No. 362611)
from BioLegend (San Diego, CA, USA), Anti-Giantin mouse monoclonal
antibody (Cat. No. ab37266) purchased from Abcam (Waltham, Boston,
USA), LAMP1 (D2D11) XP rabbit monoclonal antibody (Cat. No. 9091)
obtained from Cell Signaling Technology (Danvers, Massachusetts, USA),
and Cy3-AffiniPure F(ab’)2 Fragment Donkey Anti-Rabbit IgG
(H+L) polyclonal antibody (Cat. No. 711-166-152) and Cy3-AffiniPure
Donkey Anti-Mouse IgG (H+L) polyclonal antibody (Cat. No. 715-165-150)
supplied by Jackson ImmunoResearch (West Grove, Pennsylvania, USA).
The following reagents were obtained from commercial sources: PBS,
FBS, HEPES, NEAA, and 0.05% Trypsin-EDTA (1x) were purchased from
Gibco (Thermo Fisher Scientific Inc., Rockford, IL, USA). DMSO, penicillin/streptomycin,
BSA, DAPI, glycerol, methanol, Triton X-100, and sodium hydrogen carbonate
were obtained from Carl Roth GmbH & Co. KG (Karlsruhe, Baden-Württemberg,
Germany).d -Luciferin Firefly was provided by Biosynth (Staad,
Switzerland), and DMEM (with: 1.0 g/L of glucose, stable glutamine,
sodium pyruvate, 3.7 g/L of NaHCO3) was purchased from
PAN Biotech (Aidenbach, Bayern, Germany).dl -Threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol
(PPMP) was obtained from Sigma-Aldrich Chemie GmbH (Saint Louis, MO,
USA).
used: Alexa Fluor 647-labeled anti-6-His epitope tag (Cat. No. 362611)
from BioLegend (San Diego, CA, USA), Anti-Giantin mouse monoclonal
antibody (Cat. No. ab37266) purchased from Abcam (Waltham, Boston,
USA), LAMP1 (D2D11) XP rabbit monoclonal antibody (Cat. No. 9091)
obtained from Cell Signaling Technology (Danvers, Massachusetts, USA),
and Cy3-AffiniPure F(ab’)2 Fragment Donkey Anti-Rabbit IgG
(H+L) polyclonal antibody (Cat. No. 711-166-152) and Cy3-AffiniPure
Donkey Anti-Mouse IgG (H+L) polyclonal antibody (Cat. No. 715-165-150)
supplied by Jackson ImmunoResearch (West Grove, Pennsylvania, USA).
The following reagents were obtained from commercial sources: PBS,
FBS, HEPES, NEAA, and 0.05% Trypsin-EDTA (1x) were purchased from
Gibco (Thermo Fisher Scientific Inc., Rockford, IL, USA). DMSO, penicillin/streptomycin,
BSA, DAPI, glycerol, methanol, Triton X-100, and sodium hydrogen carbonate
were obtained from Carl Roth GmbH & Co. KG (Karlsruhe, Baden-Württemberg,
Germany).
Switzerland), and DMEM (with: 1.0 g/L of glucose, stable glutamine,
sodium pyruvate, 3.7 g/L of NaHCO3) was purchased from
PAN Biotech (Aidenbach, Bayern, Germany).
(PPMP) was obtained from Sigma-Aldrich Chemie GmbH (Saint Louis, MO,
USA).
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