Journal: Journal of visualized experiments : JoVE

Article Title: Implantation of a Cranial Window for Repeated In Vivo Imaging in Awake Mice

doi: 10.3791/62633

Figure Lengend Snippet: Table of Materials

Article Snippet: Enrofloxacin, 2.27% Bayer Xylocaine MPF with Epinephrine (1:200,000), 10 mg/mL Fresenius Kabi USA Equipment 19-Gauge Needles Move to surgery supplies BD 305186 Beveler Narishige Dental Drill Aseptico Dissecting Microscope Nikon Fiber Lite High Intensity Illuminator Dolan-Jenner Industries Germinator Fisher Scientific Isoflurane table top system with Induction Box Harvard Apparatus in company column Isoflurane Vaporizer SurgiVet Micropipette Puller Sutter Instruments Multiphoton Microscope Sutter Instruments Picospritzer Parker Rodent Hair Trimmer Wahl Stereotaxic Instrument Stoelting Ti:Sapphire Laser Coherent Water Blanket Fisher Scientific Gas Cylinder Nitrogen Matheson NI M200 Oxygen Matheson OX M250 Mouse Strain GCaMP6f B6; 129S- Gt(ROSA)26Sor tm95.1(CAGGCaMP6f)Hze /J The Jackson Laboratory Stock No: 024105 GLAST-CreER Tg(Slc1a3-cre/ERT) 1Nat/J The Jackson Laboratory Stock No: 012586 Reagent Acetone Fisher Scientific A16P4 Duralay Liquid Patterson Dental 602-8518 Duralay Powder Patterson Dental 602-7932 Krazy Glue Office Depot KG517 Loctite 401 Henkel 40140 Loctite 454 Fisher Scientific NC9194415 Tamoxifen Sigma Aldrich T5648-1G Surgery Supplies Alcohol Prep Pads Fisher Scientific Covidien 5750 Borosilicate Glass World Precision Instruments TW100F-4 Compressed Air Fisher Scientific 23-022-523 Cotton Tip Applicators Puritan 836-WC NO BINDER Cover Glass, No. 1 thickness, 3mm/5mm Warner Instruments 64-0720, 64-0700 Eye Ointment Dechra 17033-211-38 Gelfoam Ethicon 1972 Headplate Neurotar Model 1, Model 3 Iodine Prep Pads Avantor 15648-926 Isoflurane Piramal Pipette Tips Rainin 17014340 Saline (0.9% Sodium Chloride) Med Vet International RX0.9NACL-30BAC Sugi Sponge Strips Kettenbach Dental 31002 Syringe with 26-Gauge Needle BD 309625 Transfer Pipettes Fisher Scientific 13-711-9AM Surgery Tools 15° Pointed Blade Surgistar 6500 Carbide Burs SS White Dental 14717 Forceps (Large) World Precision Instruments 14099 Forceps (Small) World Precision Instruments 501764 Hemostatic forceps World Precision Instruments 501705 Holder for 15° Pointed Blade World Precision Instruments 501247 Holder for Scalpel Blades World Precision Instruments 500236 Scalpel Blades, Size 11 Integra 4-111 Scissors World Precision Instruments 503667 Viral Vector AAV1-CAG-FLEX-tdTomato Addgene 28306-AAV1 AAV1-CaMKII-0-4-Cre Addgene 105558-AAV1 Open in a separate window Once injected, leave the pipette in the brain for 4–5 min to prevent backflow.

Techniques: Microscopy, Ointment, Transferring, Saline, Plasmid Preparation

Journal: bioRxiv

Article Title: Stem cell function in vivo is supported by an alternative glycolysis endpoint

doi: 10.64898/2026.03.30.715412

Figure Lengend Snippet: A, Schematic of glycolysis termination illustrating LDHA and PDH deletion with Mx1Cre;Ldha Δ/Δ ;Pdha1 Δ mice. B-D, Peripheral blood counts. E, Bone marrow cellularity. F–I, Numbers of neutrophils, monocytes, B, and T cells. J–L, Numbers of bone marrow erythroid progenitors. M-N, Images and quantification of methylcellulose colonies from single sorted Mx1Cre;Ldha Δ/Δ ;Pdha1 Δ/Δ HSCs or controls. O-X, HSC and progenitor number in the bone marrow of Mx1Cre;Ldha Δ/Δ ;Pdha1 Δ mice. N = 6-10 mice/genotype per time point for b-l and 7-15 mice/genotype for o-x. All data represent mean ± s.d. Statistical significance was assessed with a Welch’s test ( C ) or a t-test (rest).

Article Snippet: Ldha fl/fl (Jackson Laboratory #030112) , Pdha1 fll (Jackson Laboratory #017443) , Slc16a1 fl (a gift from B. Morrison, Johns Hopkins) and Mx1cre (Jackson Laboratory #003556) mice were previously described.

Techniques:

Journal: bioRxiv

Article Title: Stem cell function in vivo is supported by an alternative glycolysis endpoint

doi: 10.64898/2026.03.30.715412

Figure Lengend Snippet: A, Schematic of combined LDH and PDH deletion. B, Immunoblot of LDHA, LDHB, and PDH in bone marrow cells following ACK lysis of red blood cells 3 weeks after deletion. C, HSC bone marrow frequency and number per two legs of mice from the indicated genotypes. D-F, Metabolomics of bone marrow cells from Mx1Cre;Ldha Δ/Δ ; Ldhb Δ/Δ ; Pdha1 τι mice and littermate controls 3 weeks after deletion. D, PCA scores plot of the hematopoietic cell metabolome E, Volcano plot of metabolite changes in hematopoietic cells. Fold change represents metabolite levels in Mx1Cre;Ldha Δ/Δ ; Ldhb Δ/Δ ; Pdha1 τι cells relative to controls. F, Levels of metabolites in central carbon metabolism. G-J, Levels of glycolytic, redox, and TCA cycle metabolites in HSC+MPP populations measured by in vivo metabolomics analysis. K-M, Results of in vivo U 13 C-glucose tracing in the indicated HSC and progenitor populations showing isotopologue abundance or fractional enrichment of the indicated metabolites. N, Respiratory ATP-linked O 2 consumption of sorted Lin - kit + HSPCs or total CD45 + bone marrow cells calculated as the difference in O 2 consumption before and after oligomycin treatment, relative to control. Each point represents a biological replicate. All data represent mean ± s.d. Statistical significance was assessed with a Mann Whitney test ( G , K-L ) or a t-test ( C , F , N ). Statistical significance for the NAD + /NADH ratio was assessed by log transformation of data followed by a t test. Statistical significance for metabolomics experiments was assessed with multiple t-tests controlling the false discovery rate at 5% with the Benjamini, Krieger, and Yekutieli method. All figures show * p<0.05, ** p<0.01, ***p<0.001.

Article Snippet: Ldha fl/fl (Jackson Laboratory #030112) , Pdha1 fll (Jackson Laboratory #017443) , Slc16a1 fl (a gift from B. Morrison, Johns Hopkins) and Mx1cre (Jackson Laboratory #003556) mice were previously described.

Techniques: Western Blot, Lysis, In Vivo, Control, MANN-WHITNEY, Transformation Assay

Journal: bioRxiv

Article Title: Stem cell function in vivo is supported by an alternative glycolysis endpoint

doi: 10.64898/2026.03.30.715412

Figure Lengend Snippet: A–I, Numbers of multipotent and restricted myeloid progenitors in the bone marrow of Mx1Cre;Ldha Δ/Δ ;Ldhb Δ/Δ ;Pdha1 Δ mice at 3 and 6 weeks after deletion. J, Images and quantification of methylcellulose colonies from single sorted Mx1Cre;Ldha Δ/Δ ; Ldhb Δ/Δ ;Pdha1 τι HSCs or controls. K, Bone marrow cellularity. L-N, Peripheral blood counts. O–Q, Numbers of erythroid progenitors. R-U, Numbers of neutrophils, monocytes, B, and T cells in Mx1Cre;Ldha Δ/Δ ;Ldhb Δ/Δ ;Pdha1 Δ mice. N = 7 mice/genotype at 3 weeks and 10-13 mice/genotype at 6 weeks after deletion. All data represent mean ± s.d. Statistical significance was assessed with a Mann-Whitney test ( O ) or a t-test (rest). All figures show * p<0.05, ** p<0.01, ***p<0.001.

Article Snippet: Ldha fl/fl (Jackson Laboratory #030112) , Pdha1 fll (Jackson Laboratory #017443) , Slc16a1 fl (a gift from B. Morrison, Johns Hopkins) and Mx1cre (Jackson Laboratory #003556) mice were previously described.

Techniques: MANN-WHITNEY

Journal: bioRxiv

Article Title: Stem cell function in vivo is supported by an alternative glycolysis endpoint

doi: 10.64898/2026.03.30.715412

Figure Lengend Snippet: Transplantation of Mx1Cre;Ldha Δ/Δ ; Ldhb Δ/Δ ;Pdha1 τι , Mx1Cre;Ldha Δ/Δ ;Pdha1 τι , or littermate control CD45.2 + donor bone marrow cells with wild type CD45.1 + CD45.2 + competitor cells into lethally irradiated CD45.1 + recipients. A, Schematic of primary and secondary transplantation experiments. B, Schematic of transplantation of undeleted donor cells followed by deletion at 6 weeks post-transplant. C–D, Donor chimerism in C blood and D bone marrow HSPCs in primary recipients (n = 12-13 for blood; n = 9-12 for BM from 3 independent experiments) from the transplant outlined in A . E–F, Donor contribution in E blood and F bone marrow HSPCs in secondary recipients (n = 8-11 for blood; n = 4-8 for BM from 3 independent experiments) from the transplant outlined in A . G–H, Peripheral blood ( G ) and BM HSPC ( H ) chimerism (n = 20–29 for blood; n = 13–24 for BM from 6 independent experiments) from the transplant outlined in B. All data represent mean ± s.d. Statistical significance was assessed with a 1-way ANOVA followed by Dunnett’s test. All figures show * p<0.05, ** p<0.01, ***p<0.001.

Article Snippet: Ldha fl/fl (Jackson Laboratory #030112) , Pdha1 fll (Jackson Laboratory #017443) , Slc16a1 fl (a gift from B. Morrison, Johns Hopkins) and Mx1cre (Jackson Laboratory #003556) mice were previously described.

Techniques: Transplantation Assay, Control, Irradiation

Journal: bioRxiv

Article Title: Stem cell function in vivo is supported by an alternative glycolysis endpoint

doi: 10.64898/2026.03.30.715412

Figure Lengend Snippet: A, Schematic of glucose-consumption and metabolite-production assays. B-C, Glucose consumption and pyruvate and lactate production in sorted Lin - Kit + HSPCs or CD45 + bone marrow cells after LDH deletion. Cells were cultured ex vivo with 0.5 mM glucose for 12 hours. depleted metabolite in sorted Mx1Cre;Ldha Δ/Δ ;Ldhb Δ/Δ Lin - Kit + HSPCs and CD45 + bone marrow cells. The x-axis shows fold change (FC) in Ldha Δ/Δ ;Ldhb Δ/Δ relative to Ldha fl/fl ;Ldhb fl/fl samples. D-E, Volcano plots showing pyruvate as the major elevated metabolite, and lactate as the major depleted metabolite in sorted Mx1Cre;Ldha D / D ;Ldhb D / D Lin-Kit+ HSPCs and CD45+ bone marrow cells. The x-axis shows fold change (FC) in Ldha D / D ;Ldhb D / D relative to Ldhafl/fl;Ldhbfl/fl samples. F–G, Glucose-derived pyruvate and lactate production in sorted HSPCs or CD45 + BM cells cultured for 12 hours with 0.5 mM U 13 C-glucose (n = 3–4 experiments). H, Pyruvate and lactate production in sorted Lin - Kit + HSPCs or CD45 + bone marrow cells after LDH/PDH deletion. Cells were cultured ex vivo with 0.2 mM glucose for 12 hours. I–J, PCA plot ( I ) and volcano plot ( J ) of metabolites in BM interstitial fluid (BM-ISF). The x-axis in J shows fold change (FC) in Ldha Δ/Δ ;Ldhb Δ/Δ ;Pdha1 Δ relative to Ldha fl/fl ;Ldhb fl/fl ;Pdha1 fl samples. K, Pyruvate is highly enriched in the BM-ISF after triple deletion and lactate is highly depleted. L, Pyruvate production in sorted Lin - Kit + HSPCs bone marrow cells cultured ex vivo with 1 mM glucose with or without 200 μM palmitate and 5 mM lactate for 12 hours. M, Schematic of BM-ISF metabolomics experiments in mice fed with high fat diet or normal chow for 1 week. N–O, Levels of pyruvate and lactate in N , BM-ISF and O serum after high fat diet or normal chow feeding. All data represent mean ± s.d. Statistical significance was assessed with a Mann-Whitney test ( C -lactate), Welch’s test ( H , K ), or a t-test (rest). All figures show * p<0.05, ** p<0.01, ***p<0.001.

Article Snippet: Ldha fl/fl (Jackson Laboratory #030112) , Pdha1 fll (Jackson Laboratory #017443) , Slc16a1 fl (a gift from B. Morrison, Johns Hopkins) and Mx1cre (Jackson Laboratory #003556) mice were previously described.

Techniques: Cell Culture, Ex Vivo, Derivative Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Stem cell function in vivo is supported by an alternative glycolysis endpoint

doi: 10.64898/2026.03.30.715412

Figure Lengend Snippet: A, Schematic of glycolysis termination illustrating conditional Ldha:Ldhb:Pdha1:Slc16a1 deletion with Mx1Cre . B–C, MCT1 inhibition with AZD3965 in chimeric transplant recipients reduces the fraction of donor Ldha Δ/Δ ;Ldhb Δ/Δ ;Pdha1 Δ HSCs, progenitors, and mature cells: paired donor chimerism of B myeloid, B cells and C BM HSPCs (n = 10 mice from 3 independent experiments). D–E, Competitive bone marrow transplantation of donor CD45.2 + Mx1Cre;Slc16a1 Δ/Δ , Mx1Cre;Ldha Δ/Δ ;Ldhb Δ/Δ ; Slc16a1 Δ/Δ , or Ldha fl/fl ;Ldhb fl/fl ; Slc16a1 fl/fl littermate control bone marrow cells with wild-type CD45.1;CD45.2 competitor cells to lethally irradiated CD45.1 recipient mice. Shown are donor chimerism in D peripheral blood at weeks 4-16 and E BM HSPCs at week 16 (n = 11–15 mice per genotype for blood; n = 12–15 for BM; three independent experiments). F–G, Competitive transplantation of Mx1Cre;Ldha Δ/Δ ;Ldhb Δ/Δ ; Pdha1 Δ ; Slc16a1 Δ/Δ or littermate control BM cells. Donor contribution to F peripheral blood at weeks 4-16 and G BM HSPCs at week 16 (n = 13 for blood; n = 12–13 for BM; three independent experiments). Data represent mean ± s.d. Statistical significance was assessed with a paired t-test ( B ), one-way ANOVA ( C, D - CD45 + , Myeloid, B cells, E ), a Brown-Forsythe ANOVA test ( D : T cells), t-test ( F – Myeloid cells), Mann-Whitney test ( F- CD45 + , B, T cells) and Welch’s test ( G ). All figures show * p<0.05, ** p<0.01, ***p<0.001.

Article Snippet: Ldha fl/fl (Jackson Laboratory #030112) , Pdha1 fll (Jackson Laboratory #017443) , Slc16a1 fl (a gift from B. Morrison, Johns Hopkins) and Mx1cre (Jackson Laboratory #003556) mice were previously described.

Techniques: Inhibition, Transplantation Assay, Control, Irradiation, MANN-WHITNEY

Journal: Molecular Medicine Reports

Article Title: Effect of selective inhibition or activation of PGE2 EP1 receptor on glomerulosclerosis

doi: 10.3892/mmr.2020.11353

Figure Lengend Snippet: Effects of blocking or stimulating the prostaglandin E2 receptor 1 on renal function. (A) Effects of SC-19220 and 17-pt-PGE2 on the BUN level in 5/6 Nx mice. (B) Effects of SC-19220 and 17-pt-PGE2 on the Cr level in 5/6 Nx mice. *P<0.05, **P<0.01. BUN, blood urea nitrogen; Cr, creatinine; 5/6 Nx, five-sixths nephrectomy; 17-pt-PGE2, 17-phenyl-trinor-prostaglandin E2 ethyl amide.

Article Snippet: Serum creatinine and urea nitrogen concentrations were determined by performing a Creatinine assay (cat. no. 1012; Exocell, Inc.) and Urea Nitrogen Assay (cat. no. 1016; Exocell, Inc.), according to the manufacturer's instructions.

Techniques: Blocking Assay