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In Vivo Optic Nerve Transection and NSC Transplant
… Three days before tissue collection, animals received intravitreal injections of cholera toxin subunit B (CTB; 2 µL, 1 µg/µL; List Biological Labs, Campbell, CA, USA) to anterogradely label RGC axons. Animals were perfused transcardially with cold PBS, followed by 4% PFA, and post fixed in 4% PFA overnight. The eyes were dissected and stored in PBS. The optic nerves were dissected and cryoprotected in 30% sucrose.
Retina and Optic Nerve Immunohistochemistry
… Primary antibodies to CTB (1:1000; List Biological Labs), …
Immunization was performed as described previously (17). Briefly, on days 1, 7, and 17, the mice were intranasally immunized with 5 μg of ovalbumin (OVA) (Sigma-Aldrich) alone or 10 μg of Alcaligenes LPS or 1 μg of cholera toxin (CT) isolated from Vibrio cholerae (List Biological Laboratories, Campbell, CA, USA) in 15 µL of PBS and administered as 7.5 µL in each nostril of mice without anesthesia. One week after the final immunization, nasal wash, bronchoalveolar lavage fluid (BALF), serum, nasal passage, NALT, cervical lymphoid nodes (CLNs), and spleen were collected as previously described (17, 18) and used for analysis.
EAE induction and Treg-specific deletion of Piezo1
Active EAE was induced in mice by immunization with MOG35–55 peptide (1 mg/ml) (MEVGWYRSPFSRVVHLYRNGK) emulsified in CFA supplemented with heat-killed Mycobacterium tuberculosis H37Ra (5 mg/ml). One hundred microliters of emulsion (EK-0111, Hooke Laboratories, Lawrence, MA) was injected subcutaneously at two sites over the flank region. In addition, 150 ng of pertussis toxin (PTX) (List Biological Laboratories, Campbell, CA) was injected intraperitoneally on the day of immunization and 48 hours later, as described previously (24, 56). …
Approximately 1 × 105 PBMCs per well were seeded in a 96-well tissue culture plate and cultured in 100 μl of RPMI-1640 supplemented with 10% FBS overnight at 37 °C and 5% CO2. havPD-1 EVs in various concentration were added to the cells at 50 μl per well. In control group, 50 μl of Atezolizumab or isotype control IgG1 were added. After 1-h incubation, 50 μl of 0.8 μg/ml Tetanus Toxoid (List labs, 191A) was added to wells. After 5-day culture, supernatant was collected and IFNγ was measured (R&D Systems, DIF50) according to manufacturer’s instructions.
For in vitro class-switching assays, cells were stimulated with 20 ng/mL interleukin (IL)-4 (R&D Systems, Minneapolis, MN, USA) and 10 µg/mL lipopolysaccharide (LPS; List Biological Laboratories, Campbell, CA, USA) for the time periods described …
cells were either unstimulated or primed with LPS for 3 h followed by the addition of oxidised phospholipids (ox-PAPC) (100 μg ml −1 ; Invivogen), pdA:dT (1 μg ml −1 ; Invivogen), flagellin (1 μg ml −1 ; Invivogen), Clostridium difficile (1 μg ml −1 ; List Biologicals) or nigericin …
Author did not specify which C. diffile was utilized; List Labs provides the following:
EAE was induced in male S1P1 -GFP signaling mice and GFP reporter mice aged 8-12 weeks. According to methods defined by Tsai et al [30, 31], mice were immunized with an emulsion containing 200 micrograms of Complete Freund’s Adjuvant (CFA), 100 micrograms of myelin oligodendrocyte glycoprotein (MOG35-55) peptide35–55, and 200 nanograms of Bordetella pertussis toxin (lot# 181236A1, List Biological Laboratories) on days zero and two. The spleen tissues were collected after eight days of immunization which marked the onset of the clinical EAE symptoms.
To induce EAE, female mice at 6–8 weeks old (n = 14 and 17 wild-type and Smek1-/+ mice, respectively) were immunized along their back with MOG35–55 (2 mg/ml) and Mycobacterium tuberculosis (8 mg/ml) dissolved in complete Freund’s adjuvant. Immediately after immunization and 2 days after immunization, mice received an intraperitoneal injection of pertussis toxin (200 ng; List Biological, Campbell, CA) in 100 μL phosphate-buffered saline (PBS). …